Cryptic Virulence and Avirulence Alleles Revealed by Controlled Sexual Recombination in Pea Aphids

Although aphids are worldwide crop pests, little is known about aphid effector genes underlying virulence and avirulence. Here we show that controlling the genetics of both aphid and host can reveal novel recombinant genotypes with previously undetected allelic variation in both virulence and avirulence functions. Clonal F₁ progeny populations were derived from reciprocal crosses and self-matings between two parental genotypes of pea aphid (Acyrthosiphon pisum) differing in virulence on a Medicago truncatula host carrying the RAP1 and RAP2 resistance genes. These populations showed Mendelian segregation consistent with aphid performance being controlled largely by a dominant virulence allele derived from only one parent. Altered segregation ratios on near-isogenic host genotypes differing in the region carrying RAP1 were indicative of additional heritable functions likely related to avirulence genes originating from both parents. Unexpectedly, some virulent F₁ progeny were recovered from selfing of an avirulent parent, suggesting a reservoir of cryptic alleles. Host chlorosis was associated with virulence, whereas necrotic hypersensitive-like response was not. No maternal inheritance was found for any of these characteristics, ruling out sex-linked, cytoplasmic, and endosymbiotic factors. Our results demonstrate the tractability of dissecting the genetic basis of pest-host resistance mechanisms and indicate that the annual sexual cycle in aphids may lead to frequent novel genotypes with both increased and decreased virulence. Availability of genomes for both pest and host can facilitate definition of cognate gene-for-gene relationships, potentially leading to selection of crop genotypes with multiple resistance traits.     

Exploration of Modulated Genetic Circuits Governing Virulence Determinants in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The expression of virulence genes in the human pathogen Staphylococcus aureus is strongly influenced by the multiple global regulators. The signal transduction cascade of these global regulators is accountable for recognizing and integrating the environmental cues to regulate the virulence regulon. While the production of virulent factors by individual global regulators are comparatively straightforward to define, auto-regulation of these global regulators and their impact on other regulators is more complex process. There are several reports on the production of virulent factors that are precisely regulated by switching processes of multiple global regulators including some prominent accessory regulators such as agr, sae and sar which allows S. aureus to coordinate the gene expression, and thus, provide organism an ability to act collectively. This review implicates the mechanisms involved in the global regulation of various virulence factors along with a comprehensive discussion on the differences between these signal transduction systems, their auto-induction and, coordination of classical and some comparatively new bacterial signal transduction systems.     

An offer you cannot refuse: down‐regulation of immunity in response to a pathogen's retaliation threat

According to the Red Queen hypothesis, hosts and pathogens are engaged in an escalating coevolutionary arms race between resistance and virulence. However, the vast majority of symbionts colonize their hosts' mucosal compartments without triggering any immune response, resulting in durable commensal associations. Here, I propose a simple extension of previous mathematical models for antagonistic coevolution in which the host can mount a delayed immune response; in response, the symbiont can change its virulence following this activation. Even though the levels of virulence in both phases are assumed to be genetically determined, this simple form of plasticity can select for commensal associations. In particular, coevolution can result in hosts that do not activate their immune response, thus preventing phenotypically plastic pathogens from switching to a higher virulence level. I argue that, from the host's point of view, this state is analogous to the mafia behaviour previously described in avian brood parasites. More importantly, this study provides a new hypothesis for the maintenance of a commensal relationship through antagonistic coevolution.     

Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

A Brucella Virulence Factor Targets Macrophages to Trigger B-cell Proliferation

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA-treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified nonmuscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homologue of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen.     

Dynamics of Meloidogyne incognita Virulence to Resistance Genes Rk and Rk2 in Cowpea

The virulence index of three Meloidogyne incognita field isolates to the resistance gene Rk in cowpea was 0%, 75%, and 120%, with the index measured as reproduction on resistant plants as a percentage of the reproduction on susceptible plants. Continuous culture of the 75% virulent isolate on susceptible tomato for more than 5 years (about 25 generations) resulted in virulence decline to about 4%. The rate of the decline in virulence was described by exponential decay, indicating the progressive loss of virulence on a susceptible host. The 120% virulent isolate declined to 90% virulence during five generations on susceptible cowpea. Following virulence decline, the two isolates were compared over 5 years in inoculated field microplots both separately and as a mixture on susceptible, gene Rk, and gene Rk² cowpea plants. At infestation of the plots, the two isolates were 1.2% and 92.0% virulent, respectively, to gene Rk and 0.2% and 8.1% virulent, respectively, to gene Rk². Virulence to gene Rk in the two isolates and in mixture increased under 5 years of continuous Rk cowpea plants to 129% to 172% and under Rk² cowpea plants to 113% to 139 % by year 5. Virulence to gene Rk² increased during continuous cropping with Rk cowpea plants to 42% to 47% and with Rk² cowpea plants to 22% to 48% by year 5. Selection of Rk²-virulence was slower in the isolate with low itt-virulence. The virulence to both genes Rk and Rk² in the mixed population was not different from that in the highly virulent isolate by year 5 of all cropping combinations. Selection of Rk²-virulence on plants with Rk, and vice versa, indicated at least partial overlap of gene specificity between Rk and Rk² with respect to selection of nematode virulence. This observation should be considered when resistance is used in cowpea rotations.     

N. elongata produces type IV pili that mediate interspecies gene transfer with N. gonorrhoeae

The genus Neisseria contains at least eight commensal and two pathogenic species. According to the Neisseria phylogenetic tree, commensals are basal to the pathogens. N. elongata, which is at the opposite end of the tree from N. gonorrhoeae, has been observed to be fimbriated, and these fimbriae are correlated with genetic competence in this organism. We tested the hypothesis that the fimbriae of N. elongata are Type IV pili (Tfp), and that Tfp functions in genetic competence. We provide evidence that the N. elongata fimbriae are indeed Tfp. Tfp, as well as the DNA Uptake Sequence (DUS), greatly enhance N. elongata DNA transformation. Tfp allows N. elongata to make intimate contact with N. gonorrhoeae and to mediate the transfer of antibiotic resistance markers between these two species. We conclude that Tfp functional for genetic competence is a trait of a commensal member of the Neisseria genus. Our findings provide a mechanism for the horizontal gene transfer that has been observed among Neisseria species.     

Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus

Myxococcus xanthus cells harbor two motility machineries, type IV pili (Tfp) and the A-engine. During reversals, the two machineries switch polarity synchronously. We present a mechanism that synchronizes this polarity switching. We identify the required for motility response regulator (RomR) as essential for A-motility. RomR localizes in a bipolar, asymmetric pattern with a large cluster at the lagging cell pole. The large RomR cluster relocates to the new lagging pole in parallel with cell reversals. Dynamic RomR localization is essential for cell reversals, suggesting that RomR relocalization induces the polarity switching of the A-engine. The analysis of RomR mutants shows that the output domain targets RomR to the poles and the receiver domain is essential for dynamic localization. The small GTPase MglA establishes correct RomR polarity, and the Frz two-component system regulates dynamic RomR localization. FrzS localizes with Tfp at the leading pole and relocates in an Frz-dependent manner to the opposite pole during reversals; FrzS and RomR localize and oscillate independently. The Frz system synchronizes these oscillations and thus the synchronous polarity switching of the motility machineries.     

A New Zebrafish Model of Oro-Intestinal Pathogen Colonization Reveals a Key Role for Adhesion in Protection by Probiotic Bacteria

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection.     

Commensal pathogens, with a focus on Streptococcus pneumoniae, and interactions with the human host

Many important pathogens have humans as their normal ecological niche where healthy carriage dominates over disease. The ability of these commensal pathogens, such as Streptococcus pneumoniae, to cause disease depends on a series of microbial factors as well as of genetic and environmental factors in the human host affecting the clearing capacity mediated by the innate and adaptive immune system. This delicate interplay between microbe and host affects not only the likelihood for a commensal pathogen to cause disease, but also disease type and disease severity.     

Virus shedding kinetics and unconventional virulence tradeoffs

Tradeoff theory, which postulates that virulence provides both transmission costs and benefits for pathogens, has become widely adopted by the scientific community. Although theoretical literature exploring virulence-tradeoffs is vast, empirical studies validating various assumptions still remain sparse. In particular, truncation of transmission duration as a cost of virulence has been difficult to quantify with robust controlled in vivo studies. We sought to fill this knowledge gap by investigating how transmission rate and duration were associated with virulence for infectious hematopoietic necrosis virus (IHNV) in rainbow trout (Oncorhynchus mykiss). Using host mortality to quantify virulence and viral shedding to quantify transmission, we found that IHNV did not conform to classical tradeoff theory. More virulent genotypes of the virus were found to have longer transmission durations due to lower recovery rates of infected hosts, but the relationship was not saturating as assumed by tradeoff theory. Furthermore, the impact of host mortality on limiting transmission duration was minimal and greatly outweighed by recovery. Transmission rate differences between high and low virulence genotypes were also small and inconsistent. Ultimately, more virulent genotypes were found to have the overall fitness advantage, and there was no apparent constraint on the evolution of increased virulence for IHNV. However, using a mathematical model parameterized with experimental data, it was found that host culling resurrected the virulence tradeoff and provided low virulence genotypes with the advantage. Human-induced or natural culling, as well as host population fragmentation, may be some of the mechanisms by which virulence diversity is maintained in nature. This work highlights the importance of considering non-classical virulence tradeoffs.     

Molecular basis for substrate recognition and septum cleavage by AtlA,the major N-acetylglucosaminidase of Enterococcus faecalis

The cleavage of septal peptidoglycan at the end of cell division facilitates the separation of the two daughter cells. The hydrolases involved in this process (called autolysins) are potentially lethal enzymes that can cause cell death; their activity, therefore, must be tightly controlled during cell growth. In Enterococcus faecalis, the N-acetylglucosaminidase AtlA plays a predominant role in cell separation. atlA mutants form long cell chains and are significantly less virulent in the zebrafish model of infection. The attenuated virulence of atlA mutants is underpinned by a limited dissemination of bacterial chains in the host organism and a more efficient uptake by phagocytes that clear the infection. AtlA has structural homologs in other important pathogens, such as Listeria monocytogenes and Salmonella typhimurium, and therefore represents an attractive model to design new inhibitors of bacterial pathogenesis. Here, we provide a 1.45 Å crystal structure of the E. faecalis AtlA catalytic domain that reveals a closed conformation of a conserved β-hairpin and a complex network of hydrogen bonds that bring two catalytic residues to the ideal distance for an inverting mechanism. Based on the model of the AtlA–substrate complex, we identify key residues critical for substrate recognition and septum cleavage during bacterial growth. We propose that this work will provide useful information for the rational design of specific inhibitors targeting this enterococcal virulence factor and its orthologs in other pathogens.     

Interactions among bacterial strains and fluke genotypes shape virulence of co-infection

Most studies of virulence of infection focus on pairwise host–parasite interactions. However, hosts are almost universally co-infected by several parasite strains and/or genotypes of the same or different species. While theory predicts that co-infection favours more virulent parasite genotypes through intensified competition for host resources, knowledge of the effects of genotype by genotype (G × G) interactions between unrelated parasite species on virulence of co-infection is limited. Here, we tested such a relationship by challenging rainbow trout with replicated bacterial strains and fluke genotypes both singly and in all possible pairwise combinations. We found that virulence (host mortality) was higher in co-infections compared with single infections. Importantly, we also found that the overall virulence was dependent on the genetic identity of the co-infecting partners so that the outcome of co-infection could not be predicted from the respective virulence of single infections. Our results imply that G × G interactions among co-infecting parasites may significantly affect host health, add to variance in parasite fitness and thus influence evolutionary dynamics and ecology of disease in unexpected ways.     

Yersinia type III effectors perturb host innate immune responses

The innate immune system is the first line of defense against invading pathogens. Innate immune cells recognize molecular patterns from the pathogen and mount a response to resolve the infection. The production of proinflammatory cytokines and reactive oxygen species, phagocytosis, and induced programmed cell death are processes initiated by innate immune cells in order to combat invading pathogens. However, pathogens have evolved various virulence mechanisms to subvert these responses. One strategy utilized by Gram-negative bacterial pathogens is the deployment of a complex machine termed the type III secretion system (T3SS). The T3SS is composed of a syringe-like needle structure and the effector proteins that are injected directly into a target host cell to disrupt a cellular response. The three human pathogenic Yersinia spp. (Y. pestis, Y. enterocolitica, and Y. pseudotuberculosis) are Gram-negative bacteria that share in common a 70 kb virulence plasmid which encodes the T3SS. Translocation of the Yersinia effector proteins (YopE, YopH, YopT, YopM, YpkA/YopO, and YopP/J) into the target host cell results in disruption of the actin cytoskeleton to inhibit phagocytosis, downregulation of proinflammatory cytokine/chemokine production, and induction of cellular apoptosis of the target cell. Over the past 25 years, studies on the Yersinia effector proteins have unveiled tremendous knowledge of how the effectors enhance Yersinia virulence. Recently, the long awaited crystal structure of YpkA has been solved providing further insights into the activation of the YpkA kinase domain. Multisite autophosphorylation by YpkA to activate its kinase domain was also shown and postulated to serve as a mechanism to bypass regulation by host phosphatases. In addition, novel Yersinia effector protein targets, such as caspase-1, and signaling pathways including activation of the inflammasome were identified. In this review, we summarize the recent discoveries made on Yersinia effector proteins and their contribution to Yersinia pathogenesis.     

Fruit flies learn to avoid odours associated with virulent infection

While learning to avoid toxic food is common in mammals and occurs in some insects, learning to avoid cues associated with infectious pathogens has received little attention. We demonstrate that Drosophila melanogaster show olfactory learning in response to infection with their virulent intestinal pathogen Pseudomonas entomophila. This pathogen was not aversive to taste when added to food. Nonetheless, flies exposed for 3 h to food laced with P. entomophila, and scented with an odorant, became subsequently less likely to choose this odorant than flies exposed to pathogen-laced food scented with another odorant. No such effect occurred after an otherwise identical treatment with an avirulent mutant of P. entomophila, indicating that the response is mediated by pathogen virulence. These results demonstrate that a virulent pathogen infection can act as an aversive unconditioned stimulus which flies can associate with food odours, and thus become less attracted to pathogen-contaminated food.     

Fitness of Virulent Meloidogyne incognita Isolates on Susceptible and Resistant Cowpea

A study of life-history traits was made to determine factors associated with the fitness of Meloidogyne incognita isolates virulent to resistance gene Rk in cowpea. Egg hatch, root penetration, egg mass production, and fecundity (eggs per egg mass) of avirulent and virulent phenotypes were compared among M. incognita isolates, isofemale lines, and single descent lines over multiple generations on resistant and susceptible cowpea. Variation (P ≤ 0.05) in both hatch and root penetration rates was found among isolates at a given generation. However, this variation was not consistent within nematode lines among generations, and there was no correlation with level of virulence, except for penetration and virulence on resistant cowpea at generation 20. Resistant and susceptible cowpea roots were penetrated at similar levels. Differences in reproductive factors on resistant plants were correlated with levels of virulence expression. In some isofemale lines, single descent lines, and isolates, lower (P ≤ 0.05) rates of egg mass production and fecundity on susceptible cowpea were associated with virulence to Rk, indicating a trade-off between reproductive fitness and virulence. Other virulent nematode lines from the same isolates did not have reduced reproductive ability on susceptible cowpea over 27 generations. Thus, virulent lineages varied in reproductive ability on susceptible cowpea, contributing to adaptation and maintenance of virulence within M. incognita populations under stabilizing selection.     

Low metabolic activity of biofilm formed by Enterococcus faecalis isolated from healthy humans and wild mallards (Anas platyrhynchos)

It is widely known that Enterococcus faecalis virulence is related to its biofilm formation. Although Enterococci are common commensal organisms of the gastrointestinal tract, the difference between commensal and pathogen strains remain unclear. In this study, we compare the biochemical profile of the biofilms formed by two groups of medical and two groups of commensal strains. The medical strains were isolated as pathogens from infections of urinary tract and other infections (wounds, pus and bedsores), and the commensal strains were taken from faeces of healthy volunteers and faeces of wild mallards (Anas platyrhynchos) living in an urban environment. The properties of biofilms formed by medical and commensal strains differed significantly. Commensal strains showed lower metabolic activity and glucose uptake and higher biofilm biomass than the medical ones. Consistent with glucose uptake experiments, we found that the glucose dehydrogenase gene was more expressed in medical strains. These results indicate that higher metabolic activity and lower protein concentration of E. faecalis cells within biofilms are formed during infections.     

Grazing protozoa and the evolution of the Escherichia coli O157:H7 Shiga toxin-encoding prophage

Humans play little role in the epidemiology of Escherichia coli O157:H7, a commensal bacterium of cattle. Why then does E. coli O157:H7 code for virulence determinants, like the Shiga toxins (Stxs), responsible for the morbidity and mortality of colonized humans? One possibility is that the virulence of these bacteria to humans is coincidental and these virulence factors evolved for and are maintained for other roles they play in the ecology of these bacteria. Here, we test the hypothesis that the carriage of the Stx-encoding prophage of E. coli O157:H7 increases the rate of survival of E. coli in the presence of grazing protozoa, Tetrahymena pyriformis. In the presence but not the absence of Tetrahymena, the carriage of the Stx-encoding prophage considerably augments the fitness of E. coli K-12 as well as clinical isolates of E. coli O157 by increasing the rate of survival of the bacteria in the food vacuoles of these ciliates. Grazing protozoa in the environment or natural host are likely to play a significant role in the ecology and maintenance of the Stx-encoding prophage of E. coli O157:H7 and may well contribute to the evolution of the virulence of these bacteria to colonize humans.     

Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.     

Effector-Triggered Immune Response in Arabidopsis thaliana Is a Quantitative Trait

We identified loci responsible for natural variation in Arabidopsis thaliana (Arabidopsis) responses to a bacterial pathogen virulence factor, HopAM1. HopAM1 is a type III effector protein secreted by the virulent Pseudomonas syringae strain Pto DC3000. Delivery of HopAM1 from disarmed Pseudomonas strains leads to local cell death, meristem chlorosis, or both, with varying intensities in different Arabidopsis accessions. These phenotypes are not associated with differences in bacterial growth restriction. We treated the two phenotypes as quantitative traits to identify host loci controlling responses to HopAM1. Genome-wide association (GWA) of 64 Arabidopsis accessions identified independent variants highly correlated with response to each phenotype. Quantitative trait locus (QTL) mapping in a recombinant inbred population between Bur-0 and Col-0 accessions revealed genetic linkage to regions distinct from the top GWA hits. Two major QTL associated with HopAM1-induced cell death were also associated with HopAM1-induced chlorosis. HopAM1-induced changes in Arabidopsis gene expression showed that rapid HopAM1-dependent cell death in Bur-0 is correlated with effector-triggered immune responses. Studies of the effect of mutations in known plant immune system genes showed, surprisingly, that both cell death and chlorosis phenotypes are enhanced by loss of EDS1, a regulatory hub in the plant immune-signaling network. Our results reveal complex genetic architecture for response to this particular type III virulence effector, in contrast to the typical monogenic control of cell death and disease resistance triggered by most type III effectors.