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This paper explores community structural factors in different low-income communities in the San Salvador, El Salvador that account for differences in the social context in which crack is used and in the HIV risk behaviors among crack users. Results suggest that both more distal (type of low-income community, level of violent crime and poverty) and proximate structural factors (type of site where drugs are used, and whether drugs are used within or outside of community of residence) influence HIV risk behaviors among drug users. Additionally, our results suggest that community structural factors influence the historical and geographic variation in drug use sites. Crack use and related sexual risk are growing problems in urban El Salvador. The prevalence of crack use in El Salvador is high (;; ). Studies have identified a 4.9% prevalence of crack use among 18 year olds , and 65% prevalence among young gang members.

In 2006, 100% of those receiving drug treatment in El Salvador received treatment for cocaine and crack. High risk sexual practices are common among crack users (;;; ). In particular, our research has identified high levels of sexual risk behavior among crack users in El Salvador including sex with multiple partners, inconsistent or non-use of condoms, and high rates of sex exchanges. Much research has shown that the sexual risk associated with crack use is dependent on the social context in which crack is used. As in other urban environments, crack is used in a variety of social settings in the San Salvador Metropolitan Area including private residences, trances (special locations in which crack is sold and used), motels, abandoned buildings or the street (; ).

These different settings are also the context of sexual risk practices such as sex exchanges for money or drugs, sex while high, and sexual victimization (; ). HIV research has increasingly called attention to the need to better understand structural factors that increase certain populations’ vulnerability to HIV infection (;; ). Structural factors have been variously defined, but often refer to factors that are outside individuals’ immediate control. Structural factors include environmental, economic or contextual characteristics that have more proximal or distal impacts on individuals’ behaviors and health outcomes (;;;; ). HIV research among drug users has explored the effects of a number of structural factors on HIV risk behaviors, from more distal community-level factors such as policing practices, crime and social disorder, and economic opportunities (;; ), to more proximate factors such as the social settings where drugs are consumed (; ).

However, the reasons distal factors affect HIV risk remain unclear because research often does not explore how these distal factors affect more proximate determinants of HIV risk. In other words, research is often segmented, looking either at the impact of distal factors on HIV risk behaviors, or on the more proximate factors such as the social contexts in which drug use and sexual risk occur without considering the relationship between these different levels. An example of the segmented approach to studying the structural determinants of HIV risk behavior is research that has attempted to explain the clustering of drug arrests and the prevalence of HIV and other blood borne diseases within disadvantaged neighborhoods. Such research has attempted to link community-level structural factors to sexual and drug risk behaviors (;; ). Some studies have examined the association of neighborhood demographic characteristics and HIV risk behaviors and failed to show significant associations. Other researchers have focused on the influence on sexual risk and drug use of more specific factors, including neighborhood physical and social disorder, and discovered significant relationships (; ). This disparity in findings can be explained by differences in the community factors measured.

More distal and unspecific factors such as poverty and race, for example, appear to have a more obscure or indirect impact on sexual and drug risk behaviors than indicators of social disorder, such as quality of life crimes. Thus, understanding the effects of broader community factors on sexual risk and drug use may require understanding different levels of conditions. To be sure, the availability of spaces for risk and prevention (e.g. Shooting galleries, drug markets and the availability of HIV prevention services) might be better predictors of differences in sexual- and drug-related risk behaviors. However, broader community factors may influence whether these spaces flourish in some communities but not in others.

Discovering how these more distal factors interact with the more proximal neighborhood context may help to explain how neighborhood disadvantage leads to greater HIV risk. Other research has focused exclusively on proximate structural factors such as the social context in which drugs are used, without considering how more distal factors may impact these settings.

A large body of research on drug use suggests that the social context in which drugs are consumed varies geographically and historically (;;;; ). Crack use sites vary from established places where crack users gather to buy, sell or use crack, and exchange sex for crack, to crack users’ residences where acquaintances gather informally to use crack (; ). The social dynamics and HIV protective and risky norms within these sites are equally variable and can affect the likelihood of HIV risky behaviors occurring in these sites (;; ). These social dynamics include, for example, whether there is a gatekeeper who controls access to the site, whether drugs are sold, and whether sex exchanges occur. For example, crack houses can be sites where individuals exchange sex for crack as well as places of sexual victimization and degradation (;; ). However, because of the gatekeeper control, crack houses can also be places where prevention materials, such as condoms, are provided.

Examination of community structural factors associated with different drug use sites has relied mainly on secondary analyses of historical data or retrospective accounts of drug users. Factors hypothesized to influence the types of places drugs are used include absent landlords and disinvestment in urban neighborhoods resulting in a proliferation of abandoned buildings used as shooting galleries, and gentrification and quality of life policing resulting in the proliferation of drug use in private residences with gatekeeper control. Despite anecdotal evidence that community-level structural factors affect drug use sites in structure, location, and social dynamics, little research has explicitly compared communities to determine which sites are more prevalent, and which structural characteristics of the community (e.g. Crime and disorder, policing practices, poverty) affect the stability, location and type of drug use sites. Thus, understanding how community-level structural factors affect the context of drug use may help explain differences in HIV risk in neighborhoods characterized by different forms of disadvantage. A second limitation of previous research on the influence of distal community-level factors on HIV risk is that it assumes a direct relationship between the neighborhood in which one lives and the HIV risks one is exposed to when this relationship may be complex or indirect. For example, even when there is ready access to drugs, people may choose to buy and use drugs outside their high risk neighborhoods of residence to avoid physical or sexual assault or police harassment (; ).

They may also buy drugs in other sites because they want to hide drug use and engagement in sex exchanges from family members or neighbors. Thus, the decision of whether or not to use drugs within one’s neighborhood of residence may not only depend on the risk characteristics of the neighborhood but also on other factors such as neighborhood monitoring, the possibility of violence, and arrest. In this paper, we examine the relationships between community-level structural factors and the more proximate context of drug use, and how these factors interact to affect HIV risk behaviors. We do this by taking a step-wise approach. First, we examine differences in structural factors among different types of low-income communities in the San Salvador Metropolitan area.

We explore the types of physical and social contexts of drug use that are more common in low income communities with different characteristics in terms of histories of formation, poverty, attitudes toward police, and level of violent crime and how the structural characteristics of the community influence patterns of drug use inside and outside the community. Finally, we examine the effects of community-level structural factors (community type, level of violent crime, attitudes toward the police, and poverty), drug use site types, and whether crack is used within or outside the community of residence on sexual risk for HIV. Study Sites Qualitative formative research was conducted in seven low-income communities that represent three distinct community types, Marginal Communities (3 communities), Asentamientos Urbanos Populares (AUPs) (1 community), and Older Central Communities (3 communities). Communities were selected because they represented the three community types of interest and because they had high levels of drug use and sales within the communities.

Additionally, it was necessary to select communities in which researchers had contacts with individuals who held leadership positions, either because they formed part of the community advisory boards, or held positions in important non-governmental or religious organizations in the communities. These contacts helped introduce the research team to others in the community and gain community buy in to the project. In at least a couple of occasions, initial community contacts or members of community boards of directors advised us not to conduct research in their communities due to fears of violence stemming from tensions between rival gangs, or gangs and community members. The three community types are distinguishable based on their histories of formation, physical layout and infrastructure, and housing types. Unlike many cities in developed countries, neighborhoods and communities in the San Salvador Metropolitan area have names and distinct boundaries universally recognized by residents living in or adjacent to the communities. The physical boundaries of many low-income communities are the main street or streets in which the entrance or entrances to the community are located. Particularly in the case of Marginal and AUP communities, there may be only one or two entrances to the community from a main road, with the rest of the houses located along narrow, pedestrian only pasajes.

Marginal Communities are typical of many of the squatter settlements found throughout the developing world. In El Salvador, these formed as migrants fled to the city to escape the 12-year Civil War that occurred between 1980 and 1992, or after natural disasters destroyed their homes. Housing was built on vacant land without any central planning or zoning and initially consisted of makeshift shelters made of scrap metal and other materials without any sanitation services, electricity, sewage or potable water. Many marginal communities subsequently have gained legal tenure of their land and housing and infrastructure improvement with the help of national and international non-governmental organizations (NGOs) (;; ). AUPs were formed as part of a government program in the 1970s through loans to low income workers employed in the formal economy provided by the Fondo Social para la Vivienda or “Social Housing Fund”.

As such, they are similar to many housing developments throughout the world that offer low-income housing to working class residents, and unlike Marginal Communities, were always legally recognized by the government. As such, AUPs, while still poor and overcrowded, have access to basic services such as sanitation, potable water, and sewage. AUPs are generally located on the outskirts of the San Salvador metropolitan area, close to the food processing and textile factories of the free trade zone. Older Central Communities are located in the historic center of San Salvador and resemble many “skid row” areas of cities in both the developing or developed world. In the past, Older Central Communities were mixed residential and commercial zones. Now they are characterized by highly transient populations of migrant workers, commercial sex workers and crack users.

Housing is often temporary, with residents renting rooms by the day or month in motels, shelters, or mesones (colonial style houses in which single rooms are rented with communal bathing and cooking facilities), or sleeping in public places such as the street or parks. The Older Central Communities are hubs of transportation with many large market places and brothels. They are also locations where social services, including AIDS Service Organizations, are concentrated. Participants and procedures We used Respondent Driven Sampling (RDS) to recruit active crack users in the San Salvador Metropolitan area.

RDS is a modified version of snowball sampling in which network data is gathered from each participant and chains of referral are monitored (; ). RDS has been shown to be highly effective in reaching hidden populations, and given long enough chains of referral the composition of the sample becomes stable (reaches equilibrium) and is reflective of the population of crack users regardless of initial seed selection.

Eligibility criteria included being 16 years or older, living in the San Salvador Metropolitan area, and having smoked crack in the last two weeks. Twenty-two seeds were selected from 7 low-income communities in which we gathered qualitative data (3 to 4 seeds per community) and represent the three different community types described above (N=420).

Seeds were identified during the qualitative phase of the study and selected based on their having large numbers or crack users within their network. At least one woman was seeded for each community. Four Salvadoran field staff, who had bachelor’s degrees in Community Psychology, conducted face-to-face surveys with participants at an AIDS Service Organization. Each seed was screened for eligibility and, after completing the interview, given three coupons to refer other crack users to the project.

All participants gave written informed consent prior to screening. Participants were paid $5 US for completing a survey and $3 for each eligible participant, maximum three, they recruited into the project.

All study procedures were approved by Institutional Review Boards at the Medical College of Wisconsin, the Universidad Centroamericana, and the Institute for Community Research. Measures In addition to basic demographic information (age, gender, level of education, and monthly income), we measured the frequency and quantity of use of alcohol and crack use in the past month. We also included the identification number of the person who referred the participant (recruitment group) in order to account for possible non-independence of participants due to the method of recruitment.

Seed participants were included with their referrals only if they referred other participants into the study. Community type captured the type of community in which participants resided which were coded on the basis of the structural characteristics of the community described above. Community types included: Marginal Communities (formed as squatter settlements); Asentamientos Urbanos Populares (AUPs) (formed with low-income loans provided by the government and located near free-trade zones); Older Central Communities (located in the historic downtown area of San Salvador and formed prior to 1950).

Another category (Other) was created for crack users who were homeless residing in commercial areas, or those who lived in middle class communities (n=48). Four independent coders coded each community for community type. Inter-rater reliability was very good (Cohen’s Kappa = 0.81, 95% CI=.76–.85). Discrepancies among coders were resolved through consensus. Community-Level Structural Factors These factors were assessed through self report as national population wide surveys in El Salvador measuring economic, employment, and demographic characteristics cannot be disaggregated beyond the municipal level.

Measures included 6 items assessing Poverty and Under-employment. Sample items are: “People in your neighborhood have work? “How many people in your neighborhood earn enough to get by?” and “How many people in your community have completed higher education (university or technical school)?” Response options were captured on a 4-point scale (1=”most” to 4= “none”, α=.84).

Attitudes toward the Police was assessed with 16 items. Sample items are: “The police are effective in combating crime in your neighborhood” and “The police view people in your neighborhood as criminals.” Response options were captured on a 4 point Likert type scale (1=”strongly agree” to 4=”strongly disagree”, α=.85). Level of Violent Crime in neighborhoods was assessed with 8 items. Sample items are “How often do shootings occur in your neighborhood” and “How often do extortions occur in your neighborhood.” Response items were captured on a 4-point Likert-type scale (1=almost never” to 4=”almost always”).

We also asked participants about the presence of gangs in their community and, if so, which gangs. Finally, we asked participants about the level of police presence in their communities including a permanent police substation, by patrol, or only when called in emergencies.

Drug Use Sites were elicited by asking participants to select from a list of options the type of place they used drugs most frequently in the last 30 days. Response options included: your own house or apartment; someone else’s house or apartment; an abandoned house/building; in the street/alley; motel; brothel; bar/club; trance (place where crack is used and sold); destroyer (abandoned house used by gangs); park/recreational area; vacant lot; or other place. These were recoded to include: private residence (own or someone else’s house); public place (abandoned building, street park/recreational area, destroyer, vacant lot); motel; trance; and other.

Data Analyses To examine whether there were structural differences between different community types, we first conducted univariate analyses (Chi square for categorical data and ANOVA for continuous variables) to determine the relationship between community type and other community-level structural characteristics (level of poverty, violent crime, and attitudes toward the police). Next, to determine the relationship between more distal community-level and proximate structural factors, we conducted univariate analyses to determine the relationship between community type and drug use sites and whether drugs were bought or used outside the community. We also conducted Chi square analyses to examine the social dynamics in each drug use site type by testing whether there were differences in gatekeeper control, whether crack was sold, whether sex exchanges occurred, whether non-transactional sex occurred, and whether condoms were present in the different drug use sites. Finally, to identify the effects of more distal and proximate structural characteristics on HIV risk, we conducted univariate Poisson regressions using community-level structural characteristics (community type, level of violent crime, level of poverty, attitudes toward the police), drug use sites and drug use location, controlling for demographic characteristics, and crack and alcohol use to predict sexual risk outcomes.

Those factors that were found to be significant at p. Community Characteristics shows self-reported differences in structural characteristics for different types of communities. There were no significant differences between level of police presence or presence of gangs among community types. However, Older Central Communities had higher levels of poverty, more negative attitudes toward the police and greater overall perceptions of violent crime in the community, followed by Marginal Communities which were intermediate on these measures, and AUP communities which showed the least poverty, more favorable attitudes toward the police and less perceived violent crime. Relationships between community type, drug use sites and drug use locations As seen in below, there were significant differences in the type of drug use site most common in each community type (p.

Type of Drug Use Site Most Common within Community Types. Residents of Marginal and AUP communities were significantly more likely to use drugs in a site located within their community of residence than outside their communities of residence. However, those residents of Marginal Communities who used drugs most frequently outside their community of residence were more likely to do so in public locations. AUP residents were equally likely to use in public locations, private residences or trances if they used outside their community of residence, compared with if they used most frequently within their community of residence.

There were also significant differences in type of location most commonly used and the social dynamics (gatekeeper control and whether crack was sold on the site) and HIV risky and protective behaviors reported by participants (sex for money or crack exchanges, non-transactional sex and the availability of condoms in the site). Sex exchanges were reported to be common in public locations, trances and motels, but condoms were only commonly available in motels. Personal factors Being female was positively associated with number of sex partners with whom a condom was not used, and number of times had sex in exchange for money without a condom, and approached significance for number of times had unprotected sex for crack. Age was associated with number of times had unprotected sex in exchange for money, and number of times had unprotected sex in exchange for crack.

Neither education nor income was associated with sexual risk behaviors in the multivariate model. More frequent alcohol use was positively associated with number of sex partners with whom a condom was not used and unprotected sex for crack exchanges, while greater crack use was associated with number of times sex was exchanged for money without a condom. Distal community-level structural factors Living in a Marginal Community (compared to living in an AUP) showed a trend toward significance and was positively associated with number of sex partners with whom a condom was not used and number of unprotected sex for crack exchanges, but negatively associated with unprotected sex for money exchanges. Living in an Other Community was positively associated with unprotected sex for money exchanges. Poverty was negatively associated with sex for money exchanges. Frequency of violent crime was positively associated with all sexual risk outcomes. Drug use site and drug use location Sex for crack exchanges were predicted by using drugs most often in a public site (compared to a private residence), using in a trance, and using drugs most often within community of residence.

In contrast, using drugs most frequently within community of residence was negatively associated with frequency of unprotected sex for money exchanges. Using most often in a trance was positively associated with number of sex partners with whom a condom was not used and unprotected sex for crack exchanges, while using most often in a motel was negatively associated with number of sexual partners with whom a condom was not used. Discussion Results from this study indicate that community-level structural factors can impact proximal structural factors such as the social context in which drugs are used. While many researchers have argued for distinguishing between more distal and more proximate structural factors (; ), in practice most HIV and other health research has focused on only one level of structural factors. In part, this is because considering all possible structural determinants of health in one model would be impossibly complex (; ).

Suggests a systematic step-wise approach for integrating different levels of structural factors into a single theoretical model. The first step is to focus on one level of structural factors while identifying potential mediating factors.

The next step is then to integrate research models and results from different levels. In this paper, we attempt to do this first by examining the relationships among more distal community-level factors (e.g. Community type, levels of violent crime, poverty and underemployment, and attitudes toward police). We then examine differences among community type and the context of drug use (i.e. The types of drug use site used and whether or not participants in different community types tend to use within or outside their community of residence). Finally, we examine the effects of more distal community-level factors, proximate factors (the types of sites and location where drugs are used), and personal characteristics into a single model to examine their independent effects on sexual risk behavior.

These multiple steps illuminate reasons for geographic differences in the context in which crack use and sexual risk occurs and thus contributes to a greater understanding of factors contributing to the sexual risk of crack users in the San Salvador Metropolitan Area. Our study confirmed that low-income communities in El Salvador are not homogenous and are thus characterized by different structural characteristics. Participants from Older Central Communities reported having higher levels of poverty and underemployment, higher rates of violent crime, and more unfavorable attitudes toward the police than residents of other community types.

Important for our study, these different types of communities were associated with differences in the sites where participants used crack within their communities, and the degree to which they chose to use crack inside or outside their communities of residence. Participants in Marginal and AUP communities were more likely to use within their communities of residence, while participants from Older Central and Other Communities were equally likely to use within or outside their communities of residence.

Our qualitative research conducted in 7 different low-income communities (3 Marginal Communities, 3 Older Central Communities and 1 AUP Community) suggest reasons for these patterns of difference. These include: the transience of the population, the level of neighborhood monitoring and cohesion; normalization of drug use; gang control of drug sales; and the presence of HIV prevention services in the community (; ).

Residents of Older Central Communities tend to be highly transient, either living on the streets or in temporary housing such as motels, shelters or mesones where rooms can be rented by the week or month. Drug sales and commercial sex work are concentrated in Older Center Communities in the center of San Salvador and these areas are also commercial and transportation hubs. Older Central Community Residents reported that they often traveled to adjacent communities to buy and use drugs if the price or quality was better. Similarly, residents of Other Communities most often are homeless and live in commercial zones with no residential housing or in middle class communities. In addition, “Other Community” residents from middle class communities may not have lived in areas where crack was sold.

They may, therefore, have been more likely to use crack in the areas outside their communities where they also bought drugs. In contrast, Marginal and AUP communities are almost exclusively residential and are often located adjacent to other communities controlled by rival gang members. Residents of both AUP and Marginal Communities report that it is difficult for outsiders to enter their communities without being assaulted, although residents themselves are relatively safe from violence because they are known to their neighbors. In addition, residents of AUPs often live on the outskirts of the San Salvador Metropolitan Area and often report fear of being robbed or assaulted by rival gangs while on public transportation (Corbett et al. Residents of AUP and Marginal Communities may have been more comfortable and perceived themselves as safer using in their communities of residence.

The type of drug use site common within the community types also differed. Drug use in private residences was common in Marginal and AUP communities, but seldom occurred in Older Central or Other Communities in which public places, particularly trances, were more often used. Residents of Marginal Communities also used trances, although less often, but residents of AUP communities seldom used trances when using in their community of residence.

Motels were not used by AUP or Marginal Community residents who used within their communities, but were used by residents of Older Central and Other Communities. Community structural factors other than community type did not predict these differences, suggesting that communities may differ in important respects not captured by our survey instruments. Public places, trances and motels may have been more commonly used among residents of Older Central and Other communities, because they were often homeless or highly transient. Thus, many residents may not have private residences in which to use and were thus forced to use in public locations or trances. In addition, there is likely little monitoring of their drug use behaviors and little community cohesion (; ). In-depth interviews from residents of these areas reported not worrying about other members of the community observing their drug use, because they lived far from family and others who knew them, and because drug use is ubiquitous in these neighborhoods. Likewise, motels are common in Older Central and some Other Communities which are both more commercial areas and locations in which commercial sex work is common.

In contrast, motels do not exist in AUP or Marginal Communities and cannot therefore be used as drug use sites within the community. Residents of Marginal and AUP communities have both the opportunity to use in private residences, and are motivated to keep their drug use private from neighbors. Crack users in in-depth interviews reported that they knew their neighbors well and tried to keep their drug use hidden from them in order to avoid gossip and shaming their family members. However, residents of Marginal Communities reported using in public places and trances located within their communities to a much greater degree than residents of AUP communities. Residents in some Marginal Communities reported that drug selling and use was normalized to a great extent and therefore did not try to hide drug use and used in public locations or trances. In addition, our qualitative interviews indicate that trances are uncommon in AUP communities because gangs control drug selling and do not allow crack users to smoke crack in their presence. Our results also suggest that different drug use sites offer different opportunities for sexual risk and protective behaviors.

Sex exchanges were extremely common in public locations, trances and motels but condoms were only readily available in motels, indicating little presence of HIV protective norms in these other drug use sites. As mentioned above, motels are places where commercial sex work is common in Older Central Communities. Older Central Communities are also the only neighborhoods with any organizations providing HIV prevention services, including condom distribution. Most of these interventions are targeted to commercial sex workers. Therefore, commercial sex workers in Older Central Communities may have established condom use norms to a much greater extent than crack users in other community types, even those who exchange sex. Further supporting this interpretation, in multivariate analyses, sex work in motels was negatively associated with unprotected sex for money exchanges while using drugs in trances and public places was associated with unprotected sex for crack exchanges.

Multivariate analyses indicated that community type was associated with sexual risk outcomes. Interestingly, while living in a Marginal Community was positively associated with number of sexual partners with whom a condom was not used and unprotected sex for crack exchanges, although not quite reaching statistical significance, it was negatively associated with unprotected sex for money exchanges. Living in an Other Community, on the other hand, was associated with unprotected sex for money exchanges.

These results support interpretations of the other analyses mentioned above regarding the influence of the relative transience of the populations, community cohesion and monitoring, and the presence of HIV prevention services in the different community types. In Marginal Communities, neighbors know one another, and commercial sex work is likely to be noticed and commented upon. They are also places where it would be difficult for clients from outside the community to enter to seek commercial sex workers, due to the closed nature of the neighborhoods, and the danger of assault faced by outsiders entering the community. The positive association with unprotected sex for crack exchanges within Marginal Communities may be due to the relative hidden nature of sex exchanges in drug use sites such as trances and public places such as abandoned buildings where condoms are seldom available. Other Communities are often commercial areas where sex work strolls are located, but are not locations where HIV prevention services for commercial sex workers are located. In contrast, Older Central Communities are locations where HIV prevention services for commercial sex workers are concentrated. Thus, sex work is likely also accompanied by a more prevalent use of condoms which are distributed free to sex workers in the area.

No such efforts have been made in other types of communities located outside the center of San Salvador, although sexual risk, particularly sex for crack exchanges, are also prevalent in these areas. Few associations were found between other community-level structural characteristics and sexual risk. However, frequency of violence was strongly associated with increased sexual risk in this study. Violence, or the threat or fear of violence, may reduce individuals’ ability to negotiate condom use, particularly in sex exchanges where sexual partners may not be well known. Many studies, including out own previous research among female commercial sex workers in El Salvador, indicates that commercial sex workers and crack users who exchange sex for crack often use condoms when they can, but are unable to when faced with sexual assault (;;; ). This is one of the first papers to attempt to study the relationship between community-level structural characteristics, proximal structural characteristics including the context of drug use, and the sexual risk behaviors of crack users. The fact that the only distal structural factors to predict sexual risk were community type and level of violent crime, however, suggests that we may not have captured the most important community-level structural factors to explain differences in the more proximate context of crack use and HIV risk.

Our interpretations of our results are largely based on ethnographic knowledge of the San Salvador Metropolitan Area, in particular differences in the transience of residents in the area, community monitoring, presence of commercial sex work strolls, presence of HIV prevention materials, and in some cases, gang control of drug sales. These interpretations suggest a number of other important characteristics to measure such as: the extent to which a neighborhood is residential, commercial or mixed; the transience of residents; community cohesion and monitoring; the presence of HIV prevention resources in the community; and presence of commercial sex work strolls.

In future research we will measure community cohesion and monitoring, presence of HIV resources as well as the other factors measured in this paper to evaluate a multi-level HIV prevention intervention. Although the relationship between more distal and proximate structural factors on HIV risk is complex, results from this study suggest areas for intervention both in El Salvador and in other countries in which sexual risk associated with drug use is common. Community characteristics influence the context of crack use, which in turn effects the possibilities for HIV risk and protective behaviors. Interventions can help promote risk reduction in drug use locations in which risk behaviors are more likely to occur and few risk reduction norms exist.

For example, many interventions have focused on changing the context of drug use by, for example, providing safe injection sites , or training gatekeepers or users of drug use sites to distribute condoms and injection materials. In El Salvador, HIV risk reduction strategies could include providing condoms to gatekeepers of trances and public places and private residences used as drug use sites. However, results from this study indicate that attention to larger community characteristics is also important. HIV prevention and treatment efforts in many developing countries have been concentrated in the centers of urban areas, in part because of limited resources (; ). In the San Salvador Metropolitan Area, HIV prevention efforts largely have been concentrated in the Older Central Communities. While in many ways this may seem a rational allocation of resources, given that commercial sex work is highly visible and concentrated in these areas, our results indicate that crack users in other communities also engage in high levels of risk.

However, our results suggest that residents from AUP and Marginal Communities are less likely to leave their communities to use drugs and may therefore be reluctant to go to other parts of the city to receive services. In addition, it may be more difficult for service organizations to enter these communities to provide services. It is necessary, therefore, to work with the existing leadership structure both to enter the communities and provide services, as well as to tailor HIV prevention programs to the particular social context of drug use and sexual risk within these communities. The prevailing wisdom of global health initiative (such as the Global Fund to Fight against AIDS, Tuberculosis, and Malaria) has been to donate to NGO’s who are thought to have better access to vulnerable populations and better perceived legitimacy than government agencies (; ). However, our research suggests that the reach of NGOs is limited and that working with a larger network of formal and informal community organizations to increase the reach of HIV prevention programs is necessary. Perhaps more problematically, HIV prevention efforts in El Salvador have targeted “risk groups” such as commercial sex workers. This again follows HIV prevention strategies in many countries.

However, as seen in this paper, residents of AUP and Marginal Communities avoid direct sex for money exchanges and may eschew the label of commercial sex worker because of concern about what neighbors may think. Interventions that attempt to professionalize and empower commercial sex workers, therefore, may not be well accepted in these communities, although sex for crack exchanges are common. This is not dissimilar to the situation for women in many developed and developing countries, who may maintain sexual relationships with multiple partners who help them economically, but who would not consider themselves to be commercial sex workers. While neighborhood cohesion and monitoring may have many positive effects, such as lowering perceptions of crime and insecurity as seen among residents of Marginal and AUP communities, they may also have negative effects in that residents of those communities were more likely to hide their drug use and sex exchanges from friends and neighbors and, thus, engage in more risky behavior. Prevention efforts must address stigmatizing attitudes toward drug use and sex exchanges.

And. BioTechnology Institute, Department of Soil, Water and Climate, University of Minnesota, Saint Paul, MN, USA The formation of symbiotic nitrogen-fixing nodules on the roots and/or stem of leguminous plants involves a complex signal exchange between both partners. Since many microorganisms are present in the soil, legumes and rhizobia must recognize and initiate communication with each other to establish symbioses. This results in the formation of nodules. Rhizobia within nodules exchange fixed nitrogen for carbon from the legume. Symbiotic relationships can become non-beneficial if one partner ceases to provide support to the other. As a result, complex signal exchange mechanisms have evolved to ensure continued, beneficial symbioses.

Proper recognition and signal exchange is also the basis for host specificity. Nodule formation always provides a fitness benefit to rhizobia, but does not always provide a fitness benefit to legumes. Therefore, legumes have evolved a mechanism to regulate the number of nodules that are formed, this is called autoregulation of nodulation.

Sequencing of many different rhizobia have revealed the presence of several secretion systems - and the Type III, Type IV, and Type VI secretion systems are known to be used by pathogens to transport effector proteins. These secretion systems are also known to have an effect on host specificity and are a determinant of overall nodule number on legumes. This review focuses on signal exchange between rhizobia and legumes, particularly focusing on the role of secretion systems involved in nodule formation and host specificity. Introduction Plants interact with many different types of microbes, and these associations can be pathogenic, mutualistic, or commensal in nature. The type of relationship between a specific microbe and plant can vary based on external factors, such as changes in environment, or due to intrinsic factors of both organisms.

Both pathogenic and mutualistic interactions are dependent on communication between host and microbe and are primarily based on signal exchange. The symbiotic relationship between rhizobia and legumes has long been a focus of study because of the nitrogen fixation that occurs during the symbiosis. This symbiosis requires the rhizobia to be in close physical proximity to the legume to allow for exchange of nutrients. Nitrogen is essential for all agricultural crops, but only legumes can access nitrogen from the atmosphere through symbiosis with rhizobia. Signal exchange between rhizobia and legumes has been studied as a potential process regulating symbiosis on non-legume plants and a mechanism by which to increase nitrogen fixation in legumes. The symbiosis between legumes and rhizobia has evolved to incorporate many different levels of signal exchange, from initial contact to senescence. Two primary reasons for this signal exchange are to distinguish between symbionts and pathogens and to ensure mutualism through the exchange of carbon and fixed nitrogen.

The line between symbiont and pathogen is not always clear, as both partners can have a fitness benefit to alter the relationship to their advantage. Symbiotic associations may shift from mutually beneficial to pathogenic or vice versa, such as in the case of the plant pathogen Argobacterium, having a common ancestral history with rhizobia. It has been suggested that rhizobia can be viewed as refined pathogens. The symbiotic relationship between rhizobia and legumes can easily turn pathogenic if the plants loses the ability to regulate the total number of nodules formed or the rhizobia form nodules that do not fix nitrogen – with the plant experiencing decreased fitness by providing too much carbon to the rhizobia (; ).

Co-evolution between rhizobia and legumes is more complex because of rhizobia selection can oscillate between pathogen and symbiont. The evolutionary arms race between pathogens and plants has long been studied.

Pathogens develop new strategies for creating infections, such as evolving secretion systems to alter the host cell. In response, plants develop new strategies for detecting pathogens, such as microbe-associated molecular patterns (MAMPs), and R genes. Sequencing of various rhizobial strains has shown the presence of secretion systems similar to those used by pathogens to transfer proteins into the hosts’ cytosol. These secretion systems include the Type III (T3SS), Type IV (T4SS), and Type VI secretion systems (T6SS; ).

The evolutionary presence of these secretion systems suggests that while rhizobia and legumes co-evolved a system allowing establishment and maintenance of a symbiosis, a relationship similar to a pathogen/plant interaction also co-evolved. This review focuses on legume–rhizobia signal exchange that occurs during nodule formation, plant mechanisms for limiting nodule number, and potential strategies used by rhizobia to overcome the plants ability to limit nodule number using the T3SS, T4SS, or T6SS. Signaling Exchange During Nodule Formation Rhizobia are free-living, soil saprophytes, prior to symbiosis with plants in the family Leguminosae. Rhizobia, once inoculated into soil, can persist at low levels in the absence of a suitable host. The plant initiates symbiosis by secreting flavonoids, which are detected by the rhizobia. Flavonoids vary by plant species and are only recognized by certain, yet specific, rhizobial species, offering the first level of symbiosis specificity.

The flavonoids diffuse across the membrane of the rhizobia and induce synthesis of the NodD protein to activate transcription of other genes involved in nodulation including nod factor (NF) production. NFs are a primary signal molecule produced by bacteria and detected by the plant to induce nodule organogenesis. Structurally NFs are lipochitooligosaccharides (LCOs) with a chitin oligomer backbone.

The nodABC genes encode for the proteins required to make the core NF structure and are conserved across all rhizobia species, except two Aeschynomene-infective species (; ). The NF core is then modified by species-specific proteins resulting in various substitutions on both the reducing and non-reducing end, including glycosylation and sulfation. These substitutions are specific for each host legume and offer another level of symbiosis specificity (; ). Many surface polysaccharides are also involved in symbiosis specificity including lipopolysaccharides (LPSs), extracellular polysaccharides (EPSs), and capsular poylsaccharides (KPSs; ).

The specific structure of LCOs is known to be important for recognition by host nod factor receptors (NFRs), which are receptor kinases containing lysin motifs (LysM; ). Leucine rich repeat receptor-like kinases (LRR-RLKs) are also involved in NF perception and signaling, which results in nodule formation. Root hair curling and crack entry are the two infection mechanisms used by rhizobia. Crack entry involves rhizobia entering through cracks at the lateral root bases or stems.

Root hair curling involves recognition of NFs, this recognition results in both calcium spiking and the curling of the root hair. This is thought to involve a change in the plant cells’ polarity, resulting in a new growing direction of the root hair tip. The infection chamber enlarges and changes into a globular apoplastic space. Next, root tip growth in switched from radial to polar tip elongation.

The continued growth of the infection thread is dependent on NF specificity as well as EPS. Both the epidermis and the cortex recognize NFs, the epidermis regulates rhizobia infection and the root cortex is responsible for nodule formation. Cortical cells develop into a nodule primordium. When the infection thread reaches the nodule primordium, the rhizobia enter into the inner cells and become encapsulated within a peri-bacteroid membrane. There are two main types of nodules, indeterminate and determinate, and this is determined by the legumes. For indeterminate nodules, cell division typically begins in the inner cortex.

Indeterminate nodules maintain a persistent meristem and form distinct zones, including rhizobia invasion, active nitrogen fixation and senescence. These zones contain rhizobia in various developmental states with the proximal zone losing the ability to reproduce. Legumes belonging to the inverted repeat-lacking clade manipulate bacterial differentiation through secretion of cysteine-rich peptides, which induce membrane permeabilization, endoreduplication, and loss of independent viability (;; ). In contrast, cell division begins in the outer cortex for determinate nodules. Determinate nodules do not have a persistent meristem and form a homogenous group of rhizobia with full viability. In mature nodules, plants exchange small carbon molecules for ammonia with the rhizobia. Another important aspect of symbiosis regulation is amino acid exchange and cycling between the plant and the rhizobia.

During symbiosis some plants secrete branched chained amino acids, into the peribacteroid space, and in return the rhizobia secrete aspartate and, in some cases, alanine. Rhizobial biosynthesis of branched chained amino acids is shut down during symbiosis, preventing the use of ammonium by rhizobia and allowing the plant to incorporate ammonium into aspartate to produce asparagine (; ). After many weeks of plant growth, nodules begin to senescence, with a maximum lifespan well-short of that of the host plant. Dark stress, water stress, defoliation, or addition of nitrate can initiate premature nodule senescence (;; ). This suggest that the plant controls the duration of the symbiosis by being able to induce nodule senescence. These external factors are thought to lead to an increase in reactive oxygen species, which initiates senescence. During nodule senescence, the host plant initiates plant cell death and some rhizobia not in the symbiosome survive this process and return to a saprotrophic state in the soil.

Plant Signaling Limits Nodule Number The symbiotic relationship between rhizobia and legumes has the potential to become pathogenic if the plant loses the ability to regulate the total number of nodules or perceives the rhizobia as a pathogen. Rhizobia will generally initiate nodule formation because a symbiotic relationship always has a fitness benefit for the rhizobia. However, if the plant forms too many nodules then there is a negative effect on vegetative growth and yield (;; ). Legumes use a process called autoregulation of nodulation (AON) to control nodule number by preventing new nodule formation. The AON is thought to involve a root-derived signal being transported to the shoot, which induces a shoot-derived signal to be transported to the root – this inhibits nodule formation. After nodule formation, the plant cell begins to produce CLV3/ESR-related (CLE) peptides. CLE peptides are thought to be the signal molecule transported from the roots to the shoot as part of the signaling pathway involved in AON (, ).

The CLE-RS2 is a post-translationally arabinosylated glycopeptide derived from the CLE domain, and if externally added CLE-RS2 sufficient to inhibit nodule formation. The CLE-peptides are recognized by LRR-RLKs (;; ). These receptors then cause a signal cascade which results in cytokinins being transported from the shoot to the root, which could act as the shoot-derived signal to suppress nodule formation. In the Lotus japonicas tml mutant, shoot-applied cytokinin does not suppress nodule formation. This implies that TML acts downstream of cytokinins, and may act directly in the root cells to suppress nodulation.

TML encodes a Klech repeat-containing F-box protein and has been hypothesized to target a protein for degradation which has a positive role in nodule formation (; ). Autoregulation of nodulation signaling is a complex process involving numerous steps, some of which are still unknown. Disruption of AON at many different steps has been shown to results in a hyper-nodulation phenotype. This suggests that the AON signaling process could be potential targets for rhizobia to disrupt, in order to increase nodule formation.

Inhibition of AON, could result in the symbiotic relationship between rhizobia and legumes becoming a pathogenic one. Bacterial Secretion Systems Bacteria use a wide variety of secretion systems to export proteins and other compounds across their membranes and cell walls.

Interaction with the external environment is vital to bacterial survival, and many different transmembrane channels have evolved independently to fulfill this need. There have been reports of up to many different secretion systems, but only the first seven have been significantly investigated. These secretion systems have evolved independently, each containing a different set of core proteins. Each secretion system itself diverged into unique subfamilies based on different functions. The T1SS, T2SS, and T5SSs are thought to simply transport proteins and compounds outside of the cell.

The T3SS, T4SS, and T6SSs contain subfamilies with the ability to transport effector proteins into the cytosol of eukaryotic cells. This is important because it allows for the direct communication with, and modification of, the eukaryotic cytosol. These three secretion systems are well-understood for their role in pathogenesis as key factors in virulence and, in some cases, symbiosis. Rhizobia Secretion Systems As discussed above, rhizobia enter into unique symbioses with eukaryotic cells, through the formation of relationship with legumes. Sequencing of rhizobia strains has shown that they typically contain multiple secretion systems. However, the presence of these systems in the bacterial genome does not mean they have a role in symbiosis.

Rhizobia surface polysaccharides (LPS) have been known to suppress plant immune responses, but the T3SS and T4SS have also been speculated to have a role in suppressing the plant immune system. The T3SS and T4SS are each sub-divided into seven families based on function and protein homology (; ). The T3SS, T4SS, and T6SSs have been identified throughout various rhizobial genera and sequence homology shows similarity between known secretion systems used by bacterial pathogens. Specifically, sequence analysis of Sinorhizobium has shown that they can contain either the T3SS, T4SS or the T6SS, but typically only have one involved in symbiosis per strain. The T3SS, T4SS, and T6SS have all been shown to be involved in symbiosis and translocate effector proteins during symbiosis. These effector proteins could potentially have a function by promoting nodule formation, disrupting AON, or suppressing the plant’s immune response during invasion.

In plant pathogens, the T3SS effectors have been shown to target and suppress the plant immune response. Deletion of a specific sub-family of the T3SS or the T4SS has been shown to reduce nodule number and affect host range specificity (; ). However, their role in symbiosis is still not very well-understood.

Type III Secretion System The T3SS is a structure composed of 20–27 different proteins, and this transporter is responsible for secretion of type III effector proteins (T3Es;; ). Approximately 50% of proteins involved in secretion system channel formation are conserved in most T3SSs. These proteins are generally found clustered in a 22–50 kb pathogenicity island.

The T3SS complex spans the bacterial inner and outer membrane as well as the hosts’ membranes and allows protein transport into the host. Regions flanking the pathogenicity island can contain genes that encode for effector proteins, but most effector genes are scattered throughout the genome. Many different variations of T3SS, with varying functions, are found throughout the kingdom of bacteria. In the literature, the T3SS is first grouped by species, and then grouped by homology. The genes encoding the rhizobial T3SSs are called rhc ( Rhizobium conserved). The rhc are further subdivided into four families based on phylogenetic analyses, Rhc-1 to Rhc-4.

Of these four families, only Rhc-I has been showed to be involved in symbiosis. The functions of the other families are still unknown.

The T3SS is among the best studied secretion systems in rhizobia due to the wide species distribution of Rhc-1 and its role in symbiosis. T3SS – Rhc-I Effect on Symbiosis Early studies of the T3SS – Rhc-1 focused on knocking out the entire system through deletions or disruption of core genes. A diverse range of rhizobial species are known to contain a functional T3SS – Rhc-1 and are listed in Table. The influence of T3SSs on nodulation can vary from positive, in which nodulation is increased, to negative, in which nodulation is reduced.

In Sinorhizobium fredii strain NGR234, the T3SS has both a positive and negative affect on multiple different legume species, but may also have a neutral phenotype, where nodulation is not affected, for example on Vigna unguiculata (;; ). Similarly, rhizobia with the T3SS – Rch-1 show host-dependent phenotypes in regard to nodulation efficiency. This could explain why the T3SS – Rch-1 is found in many genera of rhizobia, but is not ubiquitous at the strain level.

The horizontal transfer of the T3SS could be an important evolutionary driver toward symbiosis or pathogenesis between bacteria and plants. The pathogen Ralstonia solanacearum was shown to be unable to nodulate Mimosa pudica when the symbiotic plasmid of Cupriavidus taiwanensis was added, but was able to nodulate M. Pudica if the T3SS was also deleted.

This shows that the T3SS can prevent symbiosis. However, deleting the T3SS effector protein GALA7 prevented pathogenic infection of Medicago truncatula. This shows that the T3SS in R. Solanacearum is required for pathogensis. In addition, C. Taiwanensis was able to nodulate Leucaena leucocephala when the T3SS in C. Taiwanensis was deleted.

These examples show how the presence of the T3SS can restrict host range by preventing symbiosis, and could have a role in bacteria transitioning from a symbiont to a pathogen. Regulation of the T3SS – Rhc-1 Expression of the T3SS is induced by plant flavonoid recognition through production of the transcriptional activator TtsI (;; ). TtsI initiates transcription of the T3SS genes and effector proteins by binding to specific cis-elements, known as tts boxes. The number and location of tts boxes varies between species and Bradyrhizobium japonicum USDA110 is known to have 52 different tts boxes. Proteins secreted by the T3SS are found downstream of tts boxes. There is not a consensus motif for proteins secreted through the T3SS. However, the signal sequence is typically found in the first ∼15 amino acids, on the N-terminus, of translocated proteins.

In addition not all gene transcription activated by tts boxes, are effector proteins translocated through the T3SS; some can have other roles in symbiosis such as the production of rhamnose-rich polysaccharides. These rhamnose-rich polysaccharides were shown to be surface LPSs, important in nodule formation, independent of the T3SS. This suggests an interesting link between secretion systems and surface polysaccharides involved in nodule formation specificity. Proteins Secreted by the T3SS – Rhc-1 Early studies to identify proteins secreted through the T3SS focused on using flavonoids to induce expression in culture and compared the external proteins to those found in a T3SS mutant.

However, these experiments did not show translocation into the host cytosol. This led to uncertainty as to whether an identified protein was an effector protein, acting inside the plant cell. A new, high-throughput technique was used to properly identify proteins that translocate through the T3SS as well as to identify effector proteins. However, this technique did not test for effector translocation into legumes, but rather the proxy of translocation through Pseudomonas syringae pv.

Tomato DC3000 into Arabidopsis Col-O. The T3E candidates are fused to Δ79AvrRpt2, which induces a hypersensitive response (HR) in Arabidopsis. Using this technique on three different strains of S. Fredii and B. Japonicum, between 13 and 36 T3Es per strain were identified.

The T3Es can vary between species and strains, but members of the same species tend to use very similar effector proteins. Proteins secreted by the T3SS can be separated into two categories – pilus forming and effectors. Proteins involved in pilus formation are secreted through the channel to assist in forming a channel through the plants cell wall or plasma membrane. NopA, NopB, and NopX are thought to be involved in the terminal formation of the T3SS, forming a pilus that penetrates the plant’s cell wall and plasma membrane (;;, ). The other secreted proteins are thought to be effector proteins, but few of these proteins have a predicted function in planta (Table ). As shown in Table, deleting the T3SS can have a positive or negative effect on symbiosis.

The T3SS is simply the means of transport for effector proteins. Deleting the T3SS prevents effector protein transport. These effector proteins play key roles in symbiosis. Despite having a known effect on symbiosis, none of these effector proteins has been expressed in legumes. Only the effectors NopL, NopT, and NopM have all been expressed in eukaryotic cells. NopL was first shown to be phosphorylated by plant kinases.

Next, NopL was shown to interfere with mitogen-activated protein kinase (MAPK) signaling in Nicotiana tabacum. MAPK signaling is involved pathogen recognition in both basal plant defense and R-mediated resistance.

Part of the plant defensive response is the induction of HR. The plant pathogen P. Syringae uses effector proteins AvrPto and AvrPtoB to interrupt MAPK signaling by degrading the plant protein FLS2 (; ).

Overexpression of MAPK signaling in plants induces HR to prevent pathogen infections. NopL was shown to suppress cell death induced by the overexpression of MAPK signaling. NopT when expressed in N. Tabacum or Arabidopsis thaliana elicited a strong HR response and necrotic symptoms. The authors did suggest that it could function as a protease and had similarity to the effector family YopT – AvrPphB.

AvrPphB is an effector in P. Syringae and functions as an autoprotease, cleaving itself to expose a myristolation site (; ). The addition of myristoyl groups after cleavage, target AvrPphB to the cell membrane. NopT has been shown to have cysteine protease activity and may use autoproteolysis for target to cell membranes, but its role is still uncertain. NopM was shown to possess E3 ubiquitin ligase activity. Furthermore, when this ability was lost through a point mutation, the positive effects on nodule formation were also lost. Even though the function of many specific proteins has not been determined, the accumulated effect of the T3SS effector proteins can be determined through deletion of the entire secretion system.

Bradyrhizobium elkanii, containing the T3SS, but not the T3SS mutant, was shown to increase the transcription of two genes in the roots of a soybean line deficient in NF recognition. These genes, ENOD40 and NIN, are involved in early nodulation regulation.

This suggests that the T3SS effector proteins may be involved in up-regulating host genes involved in nodule formation. Further research is needed to more completely understand how these individual effectors are functioning in planta. Type IV Secretion System The T4SS-b is functionally similar to the T3SS-Rch-1 and is also involved in protein translocation, but has a separate evolutionary origin. The T4SS is generally sub-divided into three families based on function, including conjugation, DNA uptake and release, and protein translocation. These three families can use similar core proteins to form the main channel and may share sequence similarity. Properly identifying which sub-family is present in a specific strain is key. In rhizobia, the T4SS-b shares strong homology to the VirB/VirD4 subunits found in Agrobacterium.

The core structure consists of 12 proteins, VirB1-B11 and VirD4. The T4SS-b, in Agrobacterium tumefaciens, is used for translocation of both T-DNA and effector proteins (; ).

The function of the T4SS-b is well-understood because of its role in plant transformation. Agrobacterium and rhizobia are closely related, and understanding of the T4SS-b in Agrobacterium has been leveraged to better understand the T4SS-b in rhizobia. T4SS-b Effect on Symbiosis Unlike the T3SS, there is a paucity of information regarding the role of the T4SS in symbiosis. A functional T4SS-b has only been identified in three different species (Table ). Similar to the T3SS, the T4SS-b can have both a positive or negative effect on symbiosis.

In Mesorhizobium loti R7A, nodulation on Lotus corniculatus reduced, but not completely lost, when the T4SS-b was partially deleted. This same deletion allowed M. Loti R7A to gain the ability to form nodules on L. Leucocephala. Deleting the T4SS-b in Sinorhizobium meliloti KH46c resulted in approximately a 50% decrease in nodule number on M.

Truncatula A17, but did not have a significant effect on M. Truncatula F83005-5. This dual positive and negative selection could explain why only 9 of 33 S. Meliloti and 11 of 13 S.

Medicae strains were found to contain the T4SS-b. Regulation of the T4SS-b Transcription of the T4SS is controlled by a two-component response regulator VirA/VirG. VirA is a membrane bound kinase that phosphorylates VirG in response to external factors. In contrast, VirG is a transcriptional activator that binds to vir boxes. In Rhizobium these regulators are induced by flavonoids that activate VirG.

Unlike the T3SS effectors, which can be present throughout the genome, T4SS tend to be near VirG (; ). Research in A.

Tumefaciens has identified a sequence motif, a positive charged C-terminus, present on effector proteins needed for translocation. This same sequence motif is also present on the only two effector proteins identified, Msi059 and Msi061, both in M. VirD4 interacts with the positive charge signal sequence to transport the protein through the channel. VirD4, and the requirement of a more specific signal sequence, could result in more specificity in protein transport. Proteins Secreted by the T4SS-b Thus far, only two proteins have been shown to transport through the T4SS-b, Msi059, and Msi061 in M. The Msi059 showed partial protein sequence similarity to a C48 cysteine peptidase. Interestingly, the NopD T3E in S.

Fredii HH103 also was a predicted C48 cysteine peptidase. The C48 cysteine peptidase family contains the protein XopD, a T3E from the plant pathogen Xanthomonas campestris. XopD encodes an active cysteine protease, and functions in planta to target SUMO-conjugated proteins.

This interferes with the plant’s ability to regulate the expression of specific proteins. Msi061 has shared protein similarity with A. Tumefaciens effector VirF. The VirF interacts with the host Skp1 to facilitate protein degradation of effector proteins VirE2 and Vip1 to unbind the T-DNA after into the host cell (; ). Skp1 is a core component of the E3 ubiquitin ligase, which mediates protein degradation. The precise activity of Msi059 and Msi061 are still unknown, but current evidence suggests a role in changing protein expression levels in planta.

Type VI Secretion System The T6SS is among the least researched secretion system involved in protein translocation. The T6SS is known to contain different subfamilies, but the sub-families and their functions have yet to be clearly defined. The number of proteins involved in forming the core structure seem to vary and there is no known secretion signal for protein transport.

Additionally, how T6SS expression is regulated is unknown. Still, the T6SS is thought to play an important role in the virulence of multiple pathogens, like Burkholderia mallei. T6SS Effect on Symbiosis The sequence for the T6SS has been found in five different species of rhizobia, R. Leguminosarum, B. Japonicum, M. Saheli, and S. Fredii (;; ).

However, a functional T6SS, with an effect on symbiosis, has only been shown in R. In this bacterium a negative effect on symbiosis was observed, where the T6SS prevented nodulation on Pisum sativum cv. A single protein was identified that is secreted through the T6SS. Sequencing of the first 50 amino acids suggested a role in ribose transport.

The effect that ribose transport has on symbiosis is unclear. More strains containing the T6SS have been identified, but not experimentally tested for function (; ). Example of Effector Involvement in Symbiosis Most studies have focused on deleting specific genes in the core structure, instead of the effector proteins, and observing the overall phenotypic change. This is likely due to the fact that the core genes, unlike effectors, do not vary between species. Additionally, the phenotypic effect(s) of a single effector knockout might be small, again with some strains containing 36 different T3Es.

One of the most well-characterized examples of the how the T3SS functions is in S. Fredii strain USDA257.

In this case S. Fredii USDA257 is both a pathogen and a symbiont. Legumes limit nodule number, and one mechanism used is to abort nodule formation, through a process similar to HR. Fredii USDA257 strain contains NopL, which suppresses cell death through preventing MAPK signaling from inducing HR and cell death (; ). This would, in theory, increase the total number of nodules formed. Soybeans have evolved an R gene, Rfg1, capable of detecting T3Es from S. Fredii USDA257.

Rfg1 encodes a TIR-NBS-LRR disease resistance protein, which are known to recognize pathogen effectors to induce disease resistant. In soybean lines expressing Rfg1, the plant prevents nodulation by S. Fredii USDA257, but not in the T3SS knockout mutant (; ).

In addition S. Fredii USDA257 formed almost twice as many nodules on the soybean lines without the Rfg1 and the recessive rj2 genes as did the T3SS knockout mutant, on three different soybean lines. Taken together, the T3SS, including NopL, can increase nodulation in soybean.

Recognition of the T3Es, by Rfg1, results in complete prevention of nodulation. NopL restricts the plant’s ability to prevent infection and nodule formation, and rhizobia become partially pathogenic through using this strategy. The specific protein which is recognized by Rfg1, either directly or indirectly, is still not known. Though this is just one example, it is consistent with observations from other studies showing both the positive and negative effects of the T3SS as listed in Table.

This dual selection also explains why the T3SS is not found in all strains of Rhizobia. Proposed Model Most of these studies were done by deleting the entire secretion system, versus knocking out only specific effector proteins. Secretion systems are not found in all strains for any species of rhizobia. Typically, if the T3SS or T4SS has a positive effect on nodulation, then deletion of the T3SS results in ∼40–60% reduction in nodule number. This shows that secretion systems are not essential for effective nodulation. If the T3SS has a negative effect on nodulation, then knocking out the T3SS or T4SS results in a gain of function phenotype, where the strain is now able for form nodules on a host genotype that it was previously unable to nodulate effectively.

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This shows that secretion systems restrict host range. Taken together, the evidence suggests that effector proteins may act in a pathogenic manner. The function of most effector proteins are not known.

Many are predicted to modify in planta protein levels, and NopL was shown to suppress defense responses. This suggest that rhizobial effector proteins act in a pathogenic manner, similar to the function of other known bacterial effector proteins. The model we propose here (Figure ), is to demonstrate three points regarding effector proteins: (1) the role of effector proteins is strictly pathogenic, and not involved in symbiosis communication between the rhizobia and host; (2) the role of effector proteins may lead to an increase nodule number. AON is the plants system for regulating nodule number.

The mechanism of action for individual effector proteins will differ, but the unifying aspect is the increase in nodule number. This increase could be achieved through forming additional nodules or the prevention of nodule senescence; and (3) plants use R genes to recognize effector proteins. This recognition results in host defense responses, which can prevent nodulation.

This serves to establish a host range for rhizobial strains possessing effector proteins which are recognized by the host. Proposed model for the role of effector proteins in symbiosis. Rhizobia secrete nod factors, which are lipochitooligosaccharides (LCOs), and are important for nodule formation and host specificity. Surface polysaccharides are also known to be involved in determining specificity for nodule formation. These include, extracellular polysaccharides (EPSs), capsular polysaccharides (KPSs), and lipopolysaccharides (LPSs).

Legumes limit the total number of nodules formed using autoregulation of nodulation (AON). Rhizobia use effector proteins, similar to pathogens, to alter plant cells to facilitate increased nodule formation. Effectors alter the symbiotic state toward pathogenesis. In response, plants can develop R genes capable of recognizing the presence of these effector protein, either directly or indirectly. Effector recognition results in the plant initiating a defense response and preventing nodule formation. Conclusion The T3SS, T4SS, and T6SS all play an important role in nodule formation in the symbiosis between rhizobia and legumes. Many studies have shown that these secretion systems have an effect on host range.

NFs and surface polysaccharides are also known to effect symbiotic host range. These factors are important for host recognition of a symbiont versus a pathogen and facilitate infection for nodule formation.

However, pathogens use effector proteins during invasion to promote virulence, and these effectors have an effect on the pathogens host range. Thus, other factors besides host range have to be used to determine the role of secretion systems in rhizobia/plant interaction.

The T3SS, T4SS, and T6SS are all known to transport effector proteins. The predicted function of these proteins in planta, plus identifying R genes which respond to the T3SS or its effectors, strongly suggest that these secretion systems are acting in a pathogenic manner.

These secretion systems function to transport proteins from rhizobia into the plant cytosol. Once in the cytosol, they act to either increase nodulation or result in decreased nodulation through plant defense recognition.

Specific changes in planta are not yet known. Identifying how rhizobia use effector protein could have an important agricultural application. Rhizobia may be using these proteins to suppress or prevent AON, and manipulation of this regulation may lead to the development of new strategies for increasing nodule formation. These effector proteins still have not been expressed in planta, in legumes, and thus their functions remain unclear. Although several hypotheses have been postulated, the role of T3SS and T4SS are still not fully understood and warrant further research. Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments We thank Chris Staley for helpful comments. The work was supported, in part, by grant 1237993 from the National Science Foundation. Reviewed by:, Stockholm University, Sweden, Wageningen University, Netherlands Copyright © 2015 Nelson and Sadowsky. This is an open-access article distributed under the terms of the.

The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.Correspondence: Michael J. Sadowsky, BioTechnology Institute, Department of Soil, Water and Climate, University of Minnesota, 140 Gortner Laboratory, 1479 Gortner Avenue, Saint Paul, MN 55108, USA, sadowsky@umn.edu.