Virulence Traits Research Articles

Exploiting diverse chemical collections to uncover novel antifungals

The rise in drug resistance amongst pathogenic fungi, paired with the limited arsenal of antifungals available is an imminent threat to our medical system. To address this, we screened two distinct compound libraries to identify novel strategies to expand the antifungal armamentarium. The first collection wasthe RIKEN Natural Product Depository (NPDepo), which was screened for antifungal activity against four major human fungal pathogens: Candida albicans, Candida glabrata, Candida auris, and Cryptococcus neoformans. Through a prioritization pipeline, one compound, NPD6433, emerged as having broad-spectrum antifungal activity and minimal mammalian cytotoxicity. Chemical-genetic and biochemical assays demonstrated that NPD6433 inhibits the essential fungal enzyme fatty acid synthase 1 (Fas1). Treatment with NPD6433 inhibited various virulence traits in C. neoformans and C. auris, and rescued mammalian cell growth in a co-culture model with C. auris. The second compound library screened was adiversity-oriented collectionfrom Boston University. This chemical screen was focused on identifying novel molecules that enhance the activity of the widely deployed antifungal, fluconazole, against C. auris. Through this endeavour, we discovered a potent compound that enhanced fluconazole efficacy against C. auris through increasing azole intracellular accumulation. This activity was dependent on expression of the multidrug transporter geneCDR1, suggesting that this compound targets efflux mechanisms. Furthermore, this molecule significantly reduced fungal burden alone and in combination with fluconazole in a murine model of C. auris disseminated infection. Overall, this work identifies novel compounds with bioactivity against fungal pathogens, revealing important biology, and paving the way for the critical development of therapeutic strategies.

The first multi-omic interaction network in the human pathogen Candida glabrata provides novel insights into the virulence regulator, Hsp90

Candida glabrata-caused candidiasis is growing but treatments remain limited by paucity of drug targets, intrinsic azole resistance and increasing resistance to other drug classes. Drug resistance is one of numerous virulence traits regulated by the chaperone, heat shock protein 90 (Hsp90) in Candida albicans via its interactions with 5% of the genome. Hsp90 also regulates key drug resistance mechanisms in C. glabrata, but little else was known about Hsp90 in this organism. Therefore, CgHsp90 interactions were elucidated by genetic and proteomic methods. A genetic network was produced by a chemical-genetic, synthetic-sick screen on a gene-deletion library covering 16% of the genome; whilst quantitative proteomics was undertaken by tandem mass tagging on wild-type cells. Both experiments were undertaken at 30°C, 37°C and 39°C and Hsp90 was perturbed using sub-lethal concentrations of Hsp90 inhibitor. Efforts to identify Hsp90 interactors at these host-infection associated temperatures produced a genetic network of 68 genes and a protein network of 2298 proteins. Of these, 4 genes and 261 proteins interacted with Hsp90 at all three temperatures, indicating a core Hsp90 interaction network. Intriguingly, both networks had enrichment for the “antibiotic biosynthesis” GO term. Two genes, BCY1 and MCM16, were found to interact with Hsp90 at multiple temperatures in both networks. These data indicate the divergence of Hsp90 networks between C. glabrata and its close relatives and offer important targets for further research. Presented here is the first multi-omic interaction network in C. glabrata, focused on the virulence and drug resistance regulator, Hsp90.

Characterizing the Type 6 Secretion System (T6SS) and its role in the virulence of avian pathogenic Escherichia coli strain APECO18.

Avian pathogenic E. coli is the causative agent of extra-intestinal infections in birds known as colibacillosis, which can manifest as localized or systemic infections. The disease affects all stages of poultry production, resulting in economic losses that occur due to morbidity, carcass condemnation and increased mortality of the birds. APEC strains have a diverse virulence trait repertoire, which includes virulence factors involved in adherence to and invasion of the host cells, serum resistance factors, and toxins. However, the pathogenesis of APEC infections remains to be fully elucidated. The Type 6 secretion (T6SS) system has recently gained attention due to its role in the infection process and protection of bacteria from host defenses in human and animal pathogens. Previous work has shown that T6SS components are involved in the adherence to and invasion of host cells, as well as in the formation of biofilm, and intramacrophage bacterial replication. Here, we analyzed the frequency of T6SS genes hcp, impK, evpB, vasK and icmF in a collection of APEC strains and their potential role in virulence-associated phenotypes of APECO18. The T6SS genes were found to be significantly more prevalent in APEC than in fecal E. coli isolates from healthy birds. Expression of T6SS genes was analyzed in culture media and upon contact with host cells. Mutants were generated for hcp, impK, evpB, and icmF and characterized for their impact on virulence-associated phenotypes, including adherence to and invasion of host model cells, and resistance to predation by Dictyostelium discoideum. Deletion of the aforementioned genes did not significantly affect adherence and invasion capabilities of APECO18. Deletion of hcp reduced resistance of APECO18 to predation by D. discoideum, suggesting that T6SS is involved in the virulence of APECO18.

Black Fungus an Add on Epidemic to Cosvid-19 Pandemic

COVID-19 patients have lower immunosuppressive CD4+ T and CD8+ T cells and henceforth patients in intensive care units (ICU) need mechanical ventilation, henceforward they stay in hospitals. These patients have been exposed to advances in fungal co-infections. COVID-19 patients progress towards mucormycosis a black fungal infection that is deadly leading to loss of sight and hearing and eventually death. This article discusses the clinical manifestations, risk factors and emphases on virulence traits and management of black fungus.

Transposable Prophages in Leptospira: An Ancient, Now Diverse, Group Predominant in Causative Agents of Weil's Disease.

The virome associated with the corkscrew shaped bacterium Leptospira, responsible for Weil’s disease, is scarcely known, and genetic tools available for these bacteria remain limited. To reduce these two issues, potential transposable prophages were searched in Leptospiraceae genomes. The 236 predicted transposable prophages were particularly abundant in the most pathogenic leptospiral clade, being potentially involved in the acquisition of virulent traits. According to genomic similarities and phylogenies, these prophages are distantly related to known transposable phages and are organized into six groups, one of them encompassing prophages with unusual TA-TA ends. Interestingly, structural and transposition proteins reconstruct different relationships between groups, suggesting ancestral recombinations. Based on the baseplate phylogeny, two large clades emerge, with specific gene-contents and high sequence divergence reflecting their ancient origin. Despite their high divergence, the size and overall genomic organization of all prophages are very conserved, a testimony to the highly constrained nature of their genomes. Finally, similarities between these prophages and the three known non-transposable phages infecting L. biflexa, suggest gene transfer between different Caudovirales inside their leptospiral host, and the possibility to use some of the transposable prophages in that model strain.

Dynamic optimization reveals alveolar epithelial cells as key mediators of host defense in invasive aspergillosis.

Aspergillus fumigatus is an important human fungal pathogen and its conidia are constantly inhaled by humans. In immunocompromised individuals, conidia can grow out as hyphae that damage lung epithelium. The resulting invasive aspergillosis is associated with devastating mortality rates. Since infection is a race between the innate immune system and the outgrowth of A. fumigatus conidia, we use dynamic optimization to obtain insight into the recruitment and depletion of alveolar macrophages and neutrophils. Using this model, we obtain key insights into major determinants of infection outcome on host and pathogen side. On the pathogen side, we predict in silico and confirm in vitro that germination speed is an important virulence trait of fungal pathogens due to the vulnerability of conidia against host defense. On the host side, we found that epithelial cells, which have been underappreciated, play a role in fungal clearance and are potent mediators of cytokine release. Both predictions were confirmed by in vitro experiments on established cell lines as well as primary lung cells. Further, our model affirms the importance of neutrophils in invasive aspergillosis and underlines that the role of macrophages remains elusive. We expect that our model will contribute to improvement of treatment protocols by focusing on the critical components of immune response to fungi but also fungal virulence traits.

Comparative Genomics of Clinical and Environmental Isolates of Vibrio spp. of Colombia: Implications of Traits Associated with Virulence and Resistance.

There is widespread concern about the increase in cases of human and animal infections caused by pathogenic Vibrio species due to the emergence of epidemic lineages. In Colombia, active surveillance by the National Institute of Health (INS) has confirmed the presence of Vibrio; however, in routine surveillance, these isolates are not genomically characterized. This study focused on the pangenome analysis of six Vibrio species: V. parahaemolyticus, V. vulnificus, V. alginolyticus, V. fluvialis, V. diabolicus and V. furnissii to determine the genetic architectures of potentially virulent and antimicrobial resistance traits. Isolates from environmental and clinical samples were genome sequenced, assembled and annotated. The most important species in public health were further characterized by multilocus sequence typing and phylogenomics. For V. parahaemolyticus, we found the virulent ST3 and ST120 genotypes. For V. vulnificus, we identified isolates belonging to lineages 1 and 2. Virulence gene homologues between species were found even in non-pathogenic species such as V. diabolicus. Annotations related to the mobilome, integrative mobile and conjugative elements and resistance genes were obtained from environmental and clinical isolates. This study contributes genomic information to the intensified surveillance program implemented by the INS to establish potential sources of vibriosis in Colombia.

Correlation between phenotypic virulence traits and antibiotic resistance in Pseudomonas aeruginosa clinical isolates

Pseudomonas aeruginosa is a ubiquitous pathogen capable of infecting virtually all tissues and its one of the standout amongst the most hazardous microorganisms of high morbidity and mortality rates especially in debilitated patients with few successful antibiotic choices available. This pathogen regulating most virulence traits by that so-called quorum sensing (QS), a cell to cell communication system. the present study was intended to phenotypically evaluate the activity of specific virulence traits (including swarming and swimming motility, protease, pyocyanin, and biofilm production) in Pseudomonas aeruginosa clinical isolates and assess the statistical correlation between these traits and antibiotic resistance. One hundred and thirteen bacterial isolates were obtained from different clinical samples and identified as P. aeruginosa, among them, 73.4% have the ability to forming biofilm with different degrees; 59.2% were able to produce pyocyanin pigment while all isolates having the ability to make swarming and swimming motility and able to produce protease enzyme with different degrees. The isolates that produce the higher levels of the virulence traits were identified by both biochemical using Vitek2 automated system and genetically via 16s rRNA gene analysis. The statistical analysis results indicate that a positive significant correlation was found between biofilm formation and other studied virulence traits except for protease (r = 0.584: 0.324, P < 0.05) while a non-significant correlation was found between biofilm formation and protease activity (r = 0.105, P ˃ 0.05). Swimming and swarming motility have a positive significant correlation with other studied virulence traits (r = 0.613: 0.297, P < 0.05) except for protease. Pyocyanin pigment production have a positive significant correlation with other studied virulence traits (r = 0.33: 0.297, P < 0.05) except for protease. on the other hand, negative significant correlations were found between biofilm formation, swimming; and swarming motility, Pyocyanin pigment production, and the susceptibility of antibiotics (r = −0.512: −0.281, P < 0.05). Detection of such correlations in P. aeruginosa is useful for study the behavior of this pathogen and may be provide a new target for the treatment of MDR infections.

Mediation of Candida species growth and virulence by the proinflammatory cytokine IL-6

Abstract Interleukin-6 (IL-6) is a cytokine with pleiotropic effects that might also influence the virulence traits of some microorganisms, but its direct influence over Candida spp. is currently unknown. The objective of the study is to determine the influence of IL-6 (250 pg/ml) on the growth rate and biofilm formation of C. albicans, C. parapsilosis, C. krusei, C. auris, and C. guilliermondii, as well as to analyze the influence of this citokine on the expression of three virulence genes (ALS3, HSP70, SAP2), respectively on the germ tube formation ability of C. albicans. The influence of IL-6 on growth rate was assessed by incubating the fungal cells in presence of IL-6 for 48 hours and assessing the optical density of the samples at five timepoints. The biofilm production in presence of IL-6 was studied in microtiter plates, using crystal-violet assay, the intensity of biofilms being evaluated by spectrophotometry. The expression of ALS3, HSP70, and SAP2 in C. albicans was studied by RT-PCR, reported to ACT1 housekeeping gene. The germ-tube test was performed to assess the influence of IL-6 on the filamentation rate of C. albicans. All test results were normalized against control, without added IL-6. The results showed that IL-6 influences the growth rate of C. albicans, C. parapsilosis, C. krusei, C. auris, and C. guilliermondii in a time-dependent way. Also, IL-6 inhibited the formation of biofilms for C. albicans and C. guilliermondii. In C. albicans cells, IL-6 induced upregulation of ALS3 and HSP70, while it down-regulated the SAP2 gene. IL-6 did not influence the germ-tube formation in C. albicans. In conclusion, IL-6 might exert, in vitro, direct effects on the virulence traits of Candida spp., and its influence is dependent on the exposure time. Non-albicans Candida species presented particular responses to IL-6.

Inhibition of Quorum Sensing and Virulence Factors of Pseudomonas aeruginosa by Biologically Synthesized Gold and Selenium Nanoparticles.

The development of microbial resistance requires a novel approach to control microbial infection. This study implies the microbial synthesis of nanometals and assessment of their antivirulent activity against Pseudomonas aeruginosa. Streptomyces isolate S91 was isolated from soil with substantial ability for growth at high salts concentrations. The cell-free supernatant of S91was utilized for the synthesis of Au-NPs and Se-NPs. The 16S rRNA sequence analysis of Streptomyces S91 revealed that S91 had a high similarity (98.82%) to Streptomyces olivaceous. The biosynthesized Au-NPs and Se-NPs were characterized using a UV-Vis spectrophotometer, dynamic light scattering, transmission electron microscopy, energy dispersive X-ray diffraction and Fourier-transform infrared spectroscopy. The quorum sensing inhibitory (QSI) potential of Au-NPs and Se-NPs and the antivirulence activity was examined against P. aeruginosa. The QSI potential was confirmed using RT-PCR. The synthesized Au-NPs and Se-NPs were monodispersed spherical shapes with particle size of 12.2 and 67.98 nm, respectively. Au-NPs and Se-NPs eliminated QS in P. aeruginosa at a concentration range of 2.3–18.5 µg/mL for Au-NPs and 2.3–592 µg/mL for Se-NPs. In addition, Au-NPs and Se-NPs significantly inhibited QS-related virulence factors, such as pyocyanin, protease and, elastase in P. aeruginosa. At the molecular level, Au-NPs and Se-NPs significantly suppressed the relative expression of QS genes and toxins. Hence, the biosynthesized Au-NPS and Se-NPS could be substantial inhibitors of QS and virulence traits of P. aeruginosa.

Biodiversity, antibiotic resistance and virulence traits of Enterococcus species in artisanal dairy products

The presence, biodiversity, and virulence traits of enterococci were investigated in 426 artisanal dairy products. Enterococci were detected in 62.91% of the samples, which consisted of 10 diverse species. Enterococcus faecalis, Enterococcus faecium, and Enterococcus casseliflavus isolates contained all 7 virulence genes tested. Of the tested virulence genes, esp with 63.55% and cylA with 13.41% showed the highest and the lowest frequency, respectively. The frequency for efaA, asa1, gelE, ace and hyl was estimated at 62.10%, 61.80%, 61.22%, 49.56% and 31.19%, respectively. Most of E. faecalis, E. faecium, Enterococcus durans, and Enterococcus gallinarum strains contained all antibiotic resistance genes tested, as well as Tn916-like transposons. In addition, 100% of E. durans, E. casseliflavus, and E. gallinarum were resistant to vancomycin. As part of this study, 75.55% of Enterococcus isolates produced biofilm. In conclusion, artisanal dairy products can be a vehicle in transmitting multidrug-resistant and virulent enterococci to humans.

Agrobacterium tumefaciens Type IV and Type VI Secretion Systems Reside in Detergent-Resistant Membranes.

Cell membranes are not homogenous but compartmentalized into lateral microdomains, which are considered as biochemical reaction centers for various physiological processes in eukaryotes and prokaryotes. Due to their special lipid and protein composition, some of these microdomains are resistant to treatment with non-ionic detergents and can be purified as detergent-resistant membranes (DRMs). Here we report the proteome of DRMs from the Gram-negative phytopathogen Agrobacterium tumefaciens. Using label-free liquid chromatography-tandem mass spectrometry, we identified proteins enriched in DRMs isolated under normal and virulence-mimicking growth conditions. Prominent microdomain marker proteins such as the SPFH (stomatin/prohibitin/flotillin/HflKC) proteins HflK, HflC and Atu3772, along with the protease FtsH were highly enriched in DRMs isolated under any given condition. Moreover, proteins involved in cell envelope biogenesis, transport and secretion, as well as motility- and chemotaxis-associated proteins were overrepresented in DRMs. Most strikingly, we found virulence-associated proteins such as the VirA/VirG two-component system, and the membrane-spanning type IV and type VI secretion systems enriched in DRMs. Fluorescence microscopy of the cellular localization of both secretion systems and of marker proteins was in agreement with the results from the proteomics approach. These findings suggest that virulence traits are micro-compartmentalized into functional microdomains in A. tumefaciens.

An Integrated Perspective on Virulence-Associated Genes (VAGs), Antimicrobial Resistance (AMR), and Phylogenetic Clusters of Pathogenic and Non-pathogenic Avian Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is an important bacterial pathogen that causes avian colibacillosis and leads to huge economic losses in the poultry industry. Different virulence traits contribute to pathogenesis of APEC infections, and antimicrobial resistance (AMR) has also been an overwhelming issue in poultry worldwide. In the present study, we aimed to investigate and compare the presence of virulence-associated genes (VAGs), AMR, and phylogenetic group's distribution among APEC and avian fecal E. coli (AFEC) strains. E. coli from birds with colisepticemia and yolk sac infection (YSI) (APEC) plus E. coli strains from the feces of healthy birds (AFEC) were compared by the aforementioned traits. In addition, the clonal relatedness was compared using Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Although all strains were susceptible to fosfomycin, ceftriaxone, and cefixime, almost all strains (98%) were multi-drug resistant (MDR). All strains (except two) harbored at least three or more VAGs, and the virulence scores tended to be higher in pathogenic strains especially in the colisepticemic group. All phylogenetic groups were found in isolates from YSI, colisepticemia, and the feces of healthy birds; however, the frequency of phylogroups varied according to the source of the isolate. B1 and C phylogroups were statistically more likely to be found among APEC from YSI and colisepticemic E. coli groups, respectively, while phylogroup A was the most frequently occurring phylogroup among AFEC strains. Our findings also revealed that AMR and VAGs are not essentially co-evolved traits as in some instances AMR strains were more prevalent among AFEC. This reflects the divergent evolutionary pathways of resistance acquisition in pathogenic or non-pathogenic avian E. coli strains. Importantly, strains related to phylogenetic group C showed higher virulence score and AMR that requires further attention. To some extent, ERIC-PCR was able to group strains by isolation source, phylogroup, or virulence genes. Further integrated studies along with assessment of more detailed genotypic and phenotypic features could potentially lead to better understanding of virulence, resistance, and evolution of ExPEC.

Virulence Characteristics of Biofilm-Forming Acinetobacter baumannii in Clinical Isolates Using a Galleria mellonella Model.

Acinetobacter baumannii is a Gram-negative coccobacillus responsible for severe hospital-acquired infections, particularly in intensive care units (ICUs). The current study was designed to characterize the virulence traits of biofilm-forming carbapenem-resistant A. baumannii causing pneumonia in ICU patients using a Galleria mellonella model. Two hundred and thirty patients with hospital-acquired or ventilator-associated pneumonia were included in our study. Among the total isolates, A. baumannii was the most frequently isolated etiological agent in ICU patients with pneumonia (54/165, 32.7%). All A. baumannii isolates were subjected to antimicrobial susceptibility testing by the Kirby–Bauer disk diffusion method, while the minimum inhibitory concentrations of imipenem and colistin were estimated using the broth microdilution technique. The biofilm formation activity of the isolates was tested using the microtiter plate technique. Biofilm quantification showed that 61.1% (33/54) of the isolates were strong biofilm producers, while 27.7% (15/54) and 11.1% (6/54) showed moderate or weak biofilm production. By studying the prevalence of carbapenemases-encoding genes among isolates, blaOXA-23-like was positive in 88.9% of the isolates (48/54). The BlaNDM gene was found in 27.7% of the isolates (15/54 isolates). BlaOXA-23-like and blaNDM genes coexisted in 25.9% (14/54 isolates). Bap and blaPER-1 genes, the biofilm-associated genes, coexisted in 5.6% (3/54) of the isolates. For in vivo assessment of A. baumannii pathogenicity, a Galleria mellonella survival assay was used. G. mellonella survival was statistically different between moderate and poor biofilm producers (p < 0.0001). The killing effect of the strong biofilm-producing group was significantly higher than that of the moderate and poor biofilm producers (p < 0.0001 for each comparison). These findings highlight the role of biofilm formation as a powerful virulence factor for carbapenem-resistant A. baumannii that causes pneumonia in the ICU.

Phenotypic and Molecular Characterization of Commensal, Community-Acquired and Nosocomial Klebsiella spp.

Klebsiella spp. is a relevant pathogen that can present acquired resistance to almost all available antibiotics, thus representing a serious threat for public health. While most studies have been focused on isolates causing community-acquired and nosocomial infections, little is known about the commensal isolates colonizing healthy subjects. We describe the molecular identification and the phenotypic characterization of commensal Klebsiella spp. from breast milk of healthy women and faeces from healthy breast-fed infants, which were compared with isolates from community-acquired infections and from a nosocomial NICU outbreak. The phylogenetic analysis of a 454-bp sequence of the rpoB gene was useful for species identification (K. pneumoniae, K. variicola, K. quasipneumoniae, K. oxytoca, K. grimontii, K. michiganensis, Raoultella planticola and R. ornithinolytica), previously misidentified as K. pneumoniae or K. oxytoca by biochemical methods. Globally, we report that commensal strains present virulence traits (virulence genes, siderophores and biofilms) comparable to community-acquired and NICU-infective isolates, thus suggesting that the human microbiota could constitute a reservoir for infection. Isolates causing NICU outbreak were multi-drug resistant (MDR) and ESBLs producers, although an imipenem-resistant commensal MDR K. quasipneumoniae isolate was also found. A commensal K. pneumoniae strain showed a potent bacteriocin-like inhibitory activity against MDR Klebsiella isolates, thus highlighting the potential role of commensal Klebsiella spp. in health and disease.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

Plasmids do not consistently stabilize cooperation across bacteria but may promote broad pathogen host-range.

Horizontal gene transfer via plasmids could favour cooperation in bacteria, because transfer of a cooperative gene turns non-cooperative cheats into cooperators. This hypothesis has received support from theoretical, genomic and experimental analyses. In contrast, we show here, with a comparative analysis across 51 diverse species, that genes for extracellular proteins, which are likely to act as cooperative ‘public goods’, were not more likely to be carried on either: (i) plasmids compared to chromosomes; or (ii) plasmids that transfer at higher rates. Our results were supported by theoretical modelling which showed that while horizontal gene transfer can help cooperative genes initially invade a population, it has less influence on the longer-term maintenance of cooperation. Instead, we found that genes for extracellular proteins were more likely to be on plasmids when they coded for pathogenic virulence traits, in pathogenic bacteria with a broad host-range.

Genome-Wide Identification of Tomato Xylem Sap Fitness Factors for Three Plant-Pathogenic Ralstonia Species.

ABSTRACTPlant-pathogenic Ralstonia spp. colonize plant xylem and cause wilt diseases on a broad range of host plants. To identify genes that promote growth of diverse Ralstonia strains in xylem sap from tomato plants, we performed genome-scale genetic screens (random barcoded transposon mutant sequencing screens [RB-TnSeq]) in three strains spanning the genetic, geographical, and physiological range of plant-pathogenic Ralstonia: Ralstonia solanacearum IBSBF1503, Ralstonia pseudosolanacearum GMI1000, and Ralstonia syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to ex vivo xylem sap and that culture media impose foreign selective pressures. Although wild-type Ralstonia grew in sap and in rich medium with similar doubling times and to a similar carrying capacity, more genes were essential for growth in sap than in rich medium. Each strain required many genes associated with envelope remodeling and repair processes for full fitness in xylem sap. These genes were associated with peptidoglycan peptide formation (murI), secretion of periplasmic proteins (tatC), periplasmic protein folding (dsbA), synthesis of osmoregulated periplasmic glucans (mdoGH), and lipopolysaccharide (LPS) biosynthesis. Mutant strains with mutations in four genes had strong, sap-specific fitness defects in all strain backgrounds: murI, thiC, purU, and a lipoprotein (RSc2007). Many amino acid biosynthesis genes were required for fitness in both minimal medium and xylem sap. Multiple mutants with insertions in virulence regulators had gains of fitness in culture media and neutral fitness in sap. Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition.IMPORTANCE Traditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery. Here, we have adapted a genome-scale approach, random barcoded transposon mutant sequencing (RB-TnSeq), to discover fitness factors that promote growth of three related bacterial strains in a common niche, tomato xylem sap. Fitness of the wild type and mutants show that Ralstonia spp. are adapted to grow well in xylem sap from their natural host plant, tomato. Our screen identified multiple sap-specific fitness factors with roles in maintaining the bacterial envelope. These factors include putative adaptations to resist plant defenses that may include antimicrobial proteins and specialized metabolites that damage bacterial membranes.

Examination of the Global Regulon of CsrA in Xanthomonas citri subsp. citri Using Quantitative Proteomics and Other Approaches.

The RNA-binding protein CsrA is a global posttranscriptional regulator and controls many physiological processes and virulence traits. Deletion of csrA caused loss of virulence, reduced motility and production of xanthan gum and substantial increase in glycogen accumulation, as well as enhanced bacterial aggregation and cell adhesion in Xanthomonas spp. How CsrA controls these traits is poorly understood. In this study, an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis was conducted to compare the protein profile of wild-type strain Xanthomonas citri subsp. citri and the isogenic ΔcsrA strain. A total of 2,374 proteins were identified, and 284 were considered to be differentially expressed proteins (DEPS), among which 151 proteins were up-regulated and 133 were down-regulated in the ΔcsrA strain with respect to the wild-type strain. Enrichment analysis and a protein-protein interaction network analysis showed that CsrA regulates bacterial secretion systems, flagella, and xanthan gum biosynthesis. Several proteins encoded by the gumB operon were down-regulated, whereas proteins associated with flagellum assembly and the type IV secretion system were up-regulated in the ΔcsrA strain relative to the Xcc306 strain. These results were confirmed by β-glucuronidase assay or Western blot. RNA secondary structure prediction and a gel-shift assay indicated that CsrA binds to the Shine-Dalgarno sequence of virB5. In addition, the iTRAQ analysis identified 248 DEPs that were not previously identified in transcriptome analyses. Among them, CsrA regulates levels of eight regulatory proteins (ColR, GacA, GlpR, KdgR, MoxR, PilH, RecX, and YgiX), seven TonB-dependent receptors, four outer membrane proteins, and two ferric enterobactin receptors. Taken together, this study greatly expands understanding of the regulatory network of CsrA in X. citri subsp. citri.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

OhrR is a central transcriptional regulator of virulence in Dickeya zeae.

Dickeya zeae is the causal agent of rice foot rot disease. The pathogen is known to rely on a range of virulence factors, including phytotoxin zeamines, extracellular enzymes, cell motility, and biofilm, which collectively contribute to the establishment of infections. Phytotoxin zeamines play a critical role in bacterial virulence; signalling pathways and regulatory mechanisms that govern bacterial virulence remain unclear. In this study, we identified a transcriptional regulator OhrR (organic hydroperoxide reductase regulator) that is involved in the regulation of zeamine production in D. zeae EC1. The OhrR null mutant was significantly attenuated in its virulence against rice seed, potato tubers and radish roots. Phenotype analysis showed that OhrR was also involved in the regulation of other virulence traits, including the production of extracellular cellulase, biofilm formation, and swimming/swarming motility. DNA electrophoretic mobility shift assay showed that OhrR directly regulates the transcription of key virulence genes and genes encoding bis‐(3′–5′)‐cyclic dimeric guanosine monophosphate synthetases. Furthermore, OhrR positively regulates the transcription of regulatory genes slyA and fis through binding to their promoter regions. Our findings identify a key regulator of the virulence of D. zeae and add new insights into the complex regulatory network that modulates the physiology and virulence of D. zeae.