The complete genome sequence and analysis of the epsilonproteobacterium Arcobacter butzleri

Background

Arcobacter butzleri is a member of the epsilon subdivision of the Proteobacteria and a close taxonomic relative of established pathogens, such as Campylobacter jejuni and Helicobacter pylori. Here we present the complete genome sequence of the human clinical isolate, A. butzleri strain RM4018.

Methodology/Principal Findings

Arcobacter butzleri is a member of the Campylobacteraceae, but the majority of its proteome is most similar to those of Sulfuromonas denitrificans and Wolinella succinogenes, both members of the Helicobacteraceae, and those of the deep-sea vent Epsilonproteobacteria Sulfurovum and Nitratiruptor. In addition, many of the genes and pathways described here, e.g. those involved in signal transduction and sulfur metabolism, have been identified previously within the epsilon subdivision only in S. denitrificans, W. succinogenes, Sulfurovum, and/or Nitratiruptor, or are unique to the subdivision. In addition, the analyses indicated also that a substantial proportion of the A. butzleri genome is devoted to growth and survival under diverse environmental conditions, with a large number of respiration-associated proteins, signal transduction and chemotaxis proteins and proteins involved in DNA repair and adaptation. To investigate the genomic diversity of A. butzleri strains, we constructed an A. butzleri DNA microarray comprising 2238 genes from strain RM4018. Comparative genomic indexing analysis of 12 additional A. butzleri strains identified both the core genes of A. butzleri and intraspecies hypervariable regions, where <70% of the genes were present in at least two strains.

Conclusion/Significance

The presence of pathways and loci associated often with non-host-associated organisms, as well as genes associated with virulence, suggests that A. butzleri is a free-living, water-borne organism that might be classified rightfully as an emerging pathogen. The genome sequence and analyses presented in this study are an important first step in understanding the physiology and genetics of this organism, which constitutes a bridge between the environment and mammalian hosts.     

Adherence to and Invasion of Human Intestinal Cells by Arcobacter Species and Their Virulence Genotypes

The genus Arcobacter is composed of 17 species which have been isolated from various sources. Of particular interest are A. butzleri, A. cryaerophilus, and A. skirrowii, as these have been associated with human cases of diarrhea, the probable transmission routes being through the ingestion of contaminated drinking water and food. To date, only limited studies of virulence traits in this genus have been undertaken. The present study used 60 Arcobacter strains isolated from different sources, representing 16 of the 17 species of the genus, to investigate their ability to adhere to and invade the human intestinal cell line Caco-2. In addition, the presence of five putative virulence genes (ciaB, cadF, cj1349, hecA, and irgA) was screened for in these strains by PCR. All Arcobacter species except A. bivalviorum and Arcobacter sp. strain W63 adhered to Caco-2 cells, and most species (10/16) were invasive. The most invasive species were A. skirrowii, A. cryaerophilus, A. butzleri, and A. defluvii. All invasive strains were positive for ciaB (encoding a putative invasion protein). Other putative virulence genes were present in other species, i.e., A. butzleri (cadF, cj1349, irgA, and hecA), A. trophiarum (cj1349), A. ellisii (cj1349), and A. defluvii (irgA). No virulence genes were detected in strains which showed little or no invasion of Caco-2 cells. These results indicate that many Arcobacter species are potential pathogens of humans and animals.     

Arcobacter butzleri Induce Colonic,Extra-Intestinal and Systemic Inflammatory Responses in Gnotobiotic IL-10 Deficient Mice in a Strain-Dependent Manner

Background

The immunopathological impact of human Arcobacter (A.) infections is under current debate. Episodes of gastroenteritis with abdominal pain and acute or prolonged watery diarrhea were reported for A. butzleri infected patients. Whereas adhesive, invasive and cytotoxic capacities have been described for A. butzleri in vitro, only limited information is available about the immunopathogenic potential and mechanisms of infection in vivo.

Methodology/Principal Findings

Gnotobiotic IL-10^-/- mice were generated by broad-spectrum antibiotic treatment and perorally infected with the A. butzleri strains CCUG 30485 and C1 shown to be invasive in cell culture assays. Bacterial colonization capacities, clinical conditions, intestinal, extra-intestinal and systemic immune responses were monitored at day six and 16 postinfection (p.i.). Despite stable intestinal A. butzleri colonization at high loads, gnotobiotic IL-10^-/- mice were virtually unaffected and did not display any overt symptoms at either time point. Notably, A. butzleri infection induced apoptosis of colonic epithelial cells which was paralleled by increased abundance of proliferating cells. Furthermore A. butzleri infection caused a significant increase of distinct immune cell populations such as T and B cells, regulatory T cells, macrophages and monocytes in the colon which was accompanied by elevated colonic TNF, IFN-γ, nitric oxide (NO), IL-6, IL-12p70 and MCP-1 concentrations. Strikingly, A. butzleri induced extra-intestinal and systemic immune responses as indicated by higher NO concentrations in kidney and increased TNF, IFN-γ, IL-12p70 and IL-6 levels in serum samples of infected as compared to naive mice. Overall, inflammatory responses could be observed earlier in the course of infection by the CCUG 30485 as compared to the C1 strain.

Conclusion/Significance

Peroral A. butzleri infection induced not only intestinal but also extra-intestinal and systemic immune responses in gnotobiotic IL-10^-/- mice in a strain-dependent manner. These findings point towards an immunopathogenic potential of A. butzleri in vertebrate hosts.     

Highly Invasive Listeria monocytogenes Strains Have Growth and Invasion Advantages in Strain Competition

Multiple Listeria monocytogenes strains can be present in the same food sample; moreover, infection with more than one L. monocytogenes strain can also occur. In this study we investigated the impact of strain competition on the growth and in vitro virulence potential of L. monocytogenes. We identified two strong competitor strains, whose growth was not (or only slightly) influenced by the presence of other strains and two weak competitor strains, which were outcompeted by other strains. Cell contact was essential for growth inhibition. In vitro virulence assays using human intestinal epithelial Caco2 cells showed a correlation between the invasion efficiency and growth inhibition: the strong growth competitor strains showed high invasiveness. Moreover, invasion efficiency of the highly invasive strain was further increased in certain combinations by the presence of a low invasive strain. In all tested combinations, the less invasive strain was outcompeted by the higher invasive strain. Studying the effect of cell contact on in vitro virulence competition revealed a complex pattern in which the observed effects depended only partially on cell-contact suggesting that competition occurs at two different levels: i) during co-cultivation prior to infection, which might influence the expression of virulence factors, and ii) during infection, when bacterial cells compete for the host cell. In conclusion, we show that growth of L. monocytogenes can be inhibited by strains of the same species leading potentially to biased recovery during enrichment procedures. Furthermore, the presence of more than one L. monocytogenes strain in food can lead to increased infection rates due to synergistic effects on the virulence potential.     

Comparative Whole-Genome Analysis of Clinical Isolates Reveals Characteristic Architecture of Mycobacterium tuberculosis Pangenome

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.     

Accessory Genes Confer a High Replication Rate to Virulent Feline Immunodeficiency Virus

Feline immunodeficiency virus (FIV) is a lentivirus that causes AIDS in domestic cats, similar to human immunodeficiency virus (HIV)/AIDS in humans. The FIV accessory protein Vif abrogates the inhibition of infection by cat APOBEC3 restriction factors. FIV also encodes a multifunctional OrfA accessory protein that has characteristics similar to HIV Tat, Vpu, Vpr, and Nef. To examine the role of vif and orfA accessory genes in FIV replication and pathogenicity, we generated chimeras between two FIV molecular clones with divergent disease potentials: a highly pathogenic isolate that replicates rapidly in vitro and is associated with significant immunopathology in vivo, FIV-C36 (referred to here as high-virulence FIV [HV-FIV]), and a less-pathogenic strain, FIV-PPR (referred to here as low-virulence FIV [LV-FIV]). Using PCR-driven overlap extension, we produced viruses in which vif, orfA, or both genes from virulent HV-FIV replaced equivalent genes in LV-FIV. The generation of these chimeras is more straightforward in FIV than in primate lentiviruses, since FIV accessory gene open reading frames have very little overlap with other genes. All three chimeric viruses exhibited increased replication kinetics in vitro compared to the replication kinetics of LV-FIV. Chimeras containing HV-Vif or Vif/OrfA had replication rates equivalent to those of the virulent HV-FIV parental virus. Furthermore, small interfering RNA knockdown of feline APOBEC3 genes resulted in equalization of replication rates between LV-FIV and LV-FIV encoding HV-FIV Vif. These findings demonstrate that Vif-APOBEC interactions play a key role in controlling the replication and pathogenicity of this immunodeficiency-inducing virus in its native host species and that accessory genes act as mediators of lentiviral strain-specific virulence.     

The E phylogroup of Escherichia coli is highly diverse and mimics the whole E. coli species population structure

To get a global picture of the population structure of the Escherichia coli phylogroup E, encompassing the O157:H7 EHEC lineage, we analysed the whole genome of 144 strains isolated from various continents, hosts and lifestyles and representative of the phylogroup diversity. The strains possess 4331 to 5440 genes with a core genome of 2771 genes and a pangenome of 33 722 genes. The distribution of these genes among the strains shows an asymmetric U-shaped distribution. E phylogenetic strains have the largest genomes of the species, partly explained by the presence of mobile genetic elements. Sixty-eight lineages were delineated, some of them exhibiting extra-intestinal virulence genes and being virulent in the mouse sepsis model. Except for the EHEC lineages and the reference EPEC, EIEC and ETEC strains, very few strains possess intestinal virulence genes. Most of the strains were devoid of acquired resistance genes, but eight strains possessed extended-spectrum beta-lactamase genes. Human strains belong to specific lineages, some of them being virulent and antibiotic-resistant [sequence type complexes (STcs) 350 and 2064]. The E phylogroup mimics all the features of the species as a whole, a phenomenon already observed at the STc level, arguing for a fractal population structure of E. coli.     

Intestine and Environment of the Chicken as Reservoirs for Extraintestinal Pathogenic Escherichia coli Strains with Zoonotic Potential

Although research has increasingly focused on the pathogenesis of avian pathogenic Escherichia coli (APEC) infections and the “APEC pathotype” itself, little is known about the reservoirs of these bacteria. We therefore compared outbreak strains isolated from diseased chickens (n = 121) with nonoutbreak strains, including fecal E. coli strains from clinically healthy chickens (n = 211) and strains from their environment (n = 35) by determining their virulence gene profiles, phylogenetic backgrounds, responses to chicken serum, and in vivo pathogenicities in a chicken infection model. In general, by examining 46 different virulence-associated genes we were able to distinguish the three groups of avian strains, but some specific fecal and environmental isolates had a virulence gene profile that was indistinguishable from that determined for outbreak strains. In addition, a substantial number of phylogenetic EcoR group B2 strains, which are known to include potent human and animal extraintestinal pathogenic E. coli (ExPEC) strains, were identified among the APEC strains (44.5%) as well as among the fecal E. coli strains from clinically healthy chickens (23.2%). Comparably high percentages (79.2 to 89.3%) of serum-resistant strains were identified for all three groups of strains tested, bringing into question the usefulness of this phenotype as a principal marker for extraintestinal virulence. Intratracheal infection of 5-week-old chickens corroborated the pathogenicity of a number of nonoutbreak strains. Multilocus sequence typing data revealed that most strains that were virulent in chicken infection experiments belonged to sequence types that are almost exclusively associated with extraintestinal diseases not only in birds but also in humans, like septicemia, urinary tract infection, and newborn meningitis, supporting the hypothesis that not the ecohabitat but the phylogeny of E. coli strains determines virulence. These data provide strong evidence for an avian intestinal reservoir hypothesis which could be used to develop intestinal intervention strategies. These strains pose a zoonotic risk because either they could be transferred directly from birds to humans or they could serve as a genetic pool for ExPEC strains.     

Pangenome inventory of Burkholderia sensu lato,Burkholderia sensu stricto,and the Burkholderia cepacia complex reveals the uniqueness of Burkholderia catarinensis

Here the pangenome analysis of Burkholderia sensu lato (s.l.) was performed for the first time, together with an updated analysis of the pangenome of Burkholderia sensu stricto, and Burkholderia cepacia complex (Bcc) focusing on the Bcc B. catarinensis specific features of its re-sequenced genome. The pangenome of Burkholderia s.l., Burkholderia s.s., and of the Bcc was open, composed of more than 96% of accessory genes, and more than 62% of unknown genes. Functional annotations showed that secondary metabolism genes belonged to the variable portion of genomes, which might explain their production of several compounds with varied bioactivities. Taken together, this work showed the great variability and uniqueness of these genomes and revealed an underexplored unknown potential in poorly characterized genes. Regarding B. catarinensis 89^T, its genome harbors genes related to hydrolases production and plant growth promotion. This draft genome will be valuable for further investigation of its biotechnological potentials.     

What It Takes to Be a Pseudomonas aeruginosa? The Core Genome of the Opportunistic Pathogen Updated

Pseudomonas aeruginosa is an opportunistic bacterial pathogen able to thrive in highly diverse ecological niches and to infect compromised patients. Its genome exhibits a mosaic structure composed of a core genome into which accessory genes are inserted en bloc at specific sites. The size and the content of the core genome are open for debate as their estimation depends on the set of genomes considered and the pipeline of gene detection and clustering. Here, we redefined the size and the content of the core genome of P. aeruginosa from fully re-analyzed genomes of 17 reference strains. After the optimization of gene detection and clustering parameters, the core genome was defined at 5,233 orthologs, which represented ~ 88% of the average genome. Extrapolation indicated that our panel was suitable to estimate the core genome that will remain constant even if new genomes are added. The core genome contained resistance determinants to the major antibiotic families as well as most metabolic, respiratory, and virulence genes. Although some virulence genes were accessory, they often related to conserved biological functions. Long-standing prophage elements were subjected to a genetic drift to eventually display a G+C content as higher as that of the core genome. This contrasts with the low G+C content of highly conserved ribosomal genes. The conservation of metabolic and respiratory genes could guarantee the ability of the species to thrive on a variety of carbon sources for energy in aerobiosis and anaerobiosis. Virtually all the strains, of environmental or clinical origin, have the complete toolkit to become resistant to the major antipseudomonal compounds and possess basic pathogenic mechanisms to infect humans. The knowledge of the genes shared by the majority of the P. aeruginosa isolates is a prerequisite for designing effective therapeutics to combat the wide variety of human infections.     

Evidence for Selection in the Abundant Accessory Gene Content of a Prokaryote Pangenome

A pangenome is the complete set of genes (core and accessory) present in a phylogenetic clade. We hypothesize that a pangenome’s accessory gene content is structured and maintained by selection. To test this hypothesis, we interrogated the genomes of 40 Pseudomonas species for statistically significant coincident (i.e., co-occurring/avoiding) gene patterns. We found that 86.7% of common accessory genes are involved in ≥1 coincident relationship. Further, genes that co-occur and/or avoid each other—but are not vertically inherited—are more likely to share functional categories, are more likely to be simultaneously transcribed, and are more likely to produce interacting proteins, than would be expected by chance. These results are not due to coincident genes being adjacent to one another on the chromosome. Together, these findings suggest that the accessory genome is structured into sets of genes that function together within a given strain. Given the similarity of the Pseudomonas pangenome with open pangenomes of other prokaryotic species, we speculate that these results are generalizable.     

Vibrio cholerae Hemolysin Is Required for Lethality,Developmental Delay,and Intestinal Vacuolation in Caenorhabditis elegans

Background

Cholera toxin (CT) and toxin-co-regulated pili (TCP) are the major virulence factors of Vibrio cholerae O1 and O139 strains that contribute to the pathogenesis of disease during devastating cholera pandemics. However, CT and TCP negative V. cholerae strains are still able to cause severe diarrheal disease in humans through mechanisms that are not well understood.

Methodology/Principal Findings

To determine the role of other virulence factors in V. cholerae pathogenesis, we used a CT and TCP independent infection model in the nematode Caenorhabditis elegans and identified the hemolysin A (hlyA) gene as a factor responsible for animal death and developmental delay. We demonstrated a correlation between the severity of infection in the nematode and the level of hemolytic activity in the V. cholerae biotypes. At the cellular level, V. cholerae infection induces formation of vacuoles in the intestinal cells in a hlyA dependent manner, consistent with the previous in vitro observations.

Conclusions/Significance

Our data strongly suggest that HlyA is a virulence factor in C. elegans infection leading to lethality and developmental delay presumably through intestinal cytopathic changes.     

Antimicrobial resistance and virulence genes profiles of Arcobacter butzleri strains isolated from back yard chickens and retail poultry meat in Chile

This research aims to investigate the presence and pathogenic potential of Arcobacter in poultry meat samples purchased in the retail market of Valdivia (South of Chile) as well as in faecal samples from backyard chickens from rural areas around this city. The isolates obtained were identified by molecular methods. Furthermore, putative virulence genes were assessed by PCR and the antimicrobial resistance was tested by phenotypic methods. Arcobacter was present in 41·6% of the samples, with the highest value in retail poultry meat (55·7%) followed by backyard production (28·0%). Arcobacter butzleri was the most prevalent species (75·6%) followed by Arcobacter skirrowii (14·8%) and Arcobacter cryaerophilus (9·6%). An 8·5% of A. butzleri strains from meat were resistant to both ciprofloxacin and tetracycline and 6·1% were resistant to erythromycin, while none was resistant to gentamycin, unlike strains from domestic chickens, which showed no resistance. Furthermore, A. butzleri strains from chicken meat presented a higher prevalence of virulence genes than strains from domestic chickens. In fact, in this last group, some genes (hecA, hecB and irgA) were completely absent. Therefore, this study provides insight on the epidemiology of Arcobacter in Chilean poultry and suggests that under traditional breeding conditions strains are, apparently, less pathogenic and drug resistant.     

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.     

DNA Mutations Mediate Microevolution between Host-Adapted Forms of the Pathogenic Fungus Cryptococcus neoformans

The disease cryptococcosis, caused by the fungus Cryptococcus neoformans, is acquired directly from environmental exposure rather than transmitted person-to-person. One explanation for the pathogenicity of this species is that interactions with environmental predators select for virulence. However, co-incubation of C. neoformans with amoeba can cause a “switch” from the normal yeast morphology to a pseudohyphal form, enabling fungi to survive exposure to amoeba, yet conversely reducing virulence in mammalian models of cryptococcosis. Like other human pathogenic fungi, C. neoformans is capable of microevolutionary changes that influence the biology of the organism and outcome of the host-pathogen interaction. A yeast-pseudohyphal phenotypic switch also happens under in vitro conditions. Here, we demonstrate that this morphological switch, rather than being under epigenetic control, is controlled by DNA mutation since all pseudohyphal strains bear mutations within genes encoding components of the RAM pathway. High rates of isolation of pseudohyphal strains can be explained by the physical size of RAM pathway genes and a hypermutator phenotype of the strain used in phenotypic switching studies. Reversion to wild type yeast morphology in vitro or within a mammalian host can occur through different mechanisms, with one being counter-acting mutations. Infection of mice with RAM mutants reveals several outcomes: clearance of the infection, asymptomatic maintenance of the strains, or reversion to wild type forms and progression of disease. These findings demonstrate a key role of mutation events in microevolution to modulate the ability of a fungal pathogen to cause disease.     

Implementation of a novel in vitro model of infection of reconstituted human epithelium for expression of virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from catheter-related infections in Mexico

Background

Methicillin-resistant Staphylococcus aureus (MRSA) are clinically relevant pathogens that cause severe catheter-related nosocomial infections driven by several virulence factors.

Methods

We implemented a novel model of infection in vitro of reconstituted human epithelium (RHE) to analyze the expression patterns of virulence genes in 21 MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. We also determined the phenotypic and genotypic co-occurrence of antibiotic- and disinfectant-resistance traits in the S. aureus strains, which were also analysed by pulsed-field-gel electrophoresis (PFGE).

Results

In this study, MRSA strains isolated from haemodialysis catheter-related infections expressed virulence markers that mediate adhesion to, and invasion of, RHE. The most frequent pattern of expression (present in 47.6% of the strains) was as follows: fnbA, fnbB, spa, clfA, clfB, cna, bbp, ebps, eap, sdrC, sdrD, sdrE, efb, icaA, and agr. Seventy-one percent of the strains harboured the antibiotic- and disinfectant-resistance genes ermA, ermB, tet(M), tet(K), blaZ, qacA, qacB, and qacC. PFGE of the isolated MRSA revealed three identical strains and two pairs of identical strains. The strains with identical PFGE patterns showed the same phenotypes and genotypes, including the same spa type (t895), suggesting hospital personnel manipulating the haemodialysis equipment could be the source of catheter contamination.

Conclusion

These findings help define the prevalence of MRSA virulence factors in catheter-related infections. Some of the products of the expressed genes that we detected in this work may serve as potential antigens for inclusion in a vaccine for the prevention of MRSA-catheter-related infections.     

Genomic Features and Niche-Adaptation of Enterococcus faecium Strains from Korean Soybean-Fermented Foods

Certain strains of Enterococcus faecium contribute beneficially to human health and food fermentation. However, other E. faecium strains are opportunistic pathogens due to the acquisition of virulence factors and antibiotic resistance determinants. To characterize E. faecium from soybean fermentation, we sequenced the genomes of 10 E. faecium strains from Korean soybean-fermented foods and analyzed their genomes by comparing them with 51 clinical and 52 non-clinical strains of different origins. Hierarchical clustering based on 13,820 orthologous genes from all E. faecium genomes showed that the 10 strains are distinguished from most of the clinical strains. Like non-clinical strains, their genomes are significantly smaller than clinical strains due to fewer accessory genes associated with antibiotic resistance, virulence, and mobile genetic elements. Moreover, we identified niche-associated gene gain and loss from the soybean strains. Thus, we conclude that soybean E. faecium strains might have evolved to have distinctive genomic features that may contribute to its ability to thrive during soybean fermentation.     

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.