Virulence-associated Traits Research Articles

Identification of Pathogen Genomic Differences That Impact Human Immune Response and Disease during Cryptococcus neoformans Infection.

Patient outcomes during infection are due to a complex interplay between the quality of medical care, host immunity factors, and the infecting pathogen's characteristics. To probe the influence of pathogen genotype on human survival, immune response, and other parameters of disease, we examined Cryptococcus neoformans isolates collected during the Cryptococcal Optimal Antiretroviral Therapy (ART) Timing (COAT) Trial in Uganda. We measured human participants' survival, meningitis disease parameters, immunologic phenotypes, and pathogen in vitro growth characteristics. We compared those clinical data to whole-genome sequences from 38 C. neoformans isolates of the most frequently observed sequence type (ST), ST93, in our Ugandan participant population and to sequences from an additional 18 strains of 9 other sequence types representing the known genetic diversity within the Ugandan Cryptococcus clinical isolates. We focused our analyses on 652 polymorphisms that were variable among the ST93 genomes, were not in centromeres or extreme telomeres, and were predicted to have a fitness effect. Logistic regression and principal component analysis identified 40 candidate Cryptococcus genes and 3 hypothetical RNAs associated with human survival, immunologic response, or clinical parameters. We infected mice with 17 available KN99α gene deletion strains for these candidate genes and found that 35% (6/17) directly influenced murine survival. Four of the six gene deletions that impacted murine survival were novel. Such bedside-to-bench translational research identifies important candidate genes for future studies on virulence-associated traits in human Cryptococcus infections.IMPORTANCE Even with the best available care, mortality rates in cryptococcal meningitis range from 20% to 60%. Disease is often due to infection by the fungus Cryptococcus neoformans and involves a complex interaction between the human host and the fungal pathogen. Although previous studies have suggested genetic differences in the pathogen impact human disease, it has proven quite difficult to identify the specific C. neoformans genes that impact the outcome of the human infection. Here, we take advantage of a Ugandan patient cohort infected with closely related C. neoformans strains to examine the role of pathogen genetic variants on several human disease characteristics. Using a pathogen whole-genome sequencing approach, we showed that 40 C. neoformans genes are associated with human disease. Surprisingly, many of these genes are specific to Cryptococcus and have unknown functions. We also show deletion of some of these genes alters disease in a mouse model of infection, confirming their role in disease. These findings are particularly important because they are the first to identify C. neoformans genes associated with human cryptococcal meningitis and lay the foundation for future studies that may lead to new treatment strategies aimed at reducing patient mortality.

Two Pseudomonas aeruginosa clonal groups belonging to the PA14 clade are indigenous to the Churince system in Cuatro Ciénegas Coahuila, México.

Pseudomonas aeruginosa is a widely distributed environmental bacterium but is also an opportunistic pathogen that represents an important health hazard due to its high intrinsic antibiotic resistance and its production of virulence factors. The genetic structure of P. aeruginosa populations using whole genome sequences shows the existence of three clades, one of which (PA7 clade) has a higher genetic diversity. These three clades include clinical and environmental isolates that are very diverse in terms of geographical origins and isolation date. Here, we report the characterization of two distinct clonal P. aeruginosa groups that form a part of the PA14 clade (clade 2) sampled from the Churince system in Cuatro Ciénegas Basin (CCB). One of the clonal groups that we report here was isolated in 2011 (group 2A) and was displaced by the other clonal group (2B) in 2015. Both Churince groups are unable to produce pyoverdine but can produce other virulence-associated traits. The existence of these unique P. aeruginosa clonal groups in the Churince system is of ecological and evolutionary significance since the microbiota of this site is generally very distinct from other lineages, and this is the first time that a population of P. aeruginosa has been found in CCB.

The Leucine-Responsive Regulatory Protein Lrp Participates in Virulence Regulation Downstream of Small RNA ArcZ in Erwinia amylovora.

Erwinia amylovora causes the devastating fire blight disease of apple and pear trees. During systemic infection of host trees, pathogen cells must rapidly respond to changes in their environment as they move through different host tissues that present distinct challenges and sources of nutrition. Growing evidence indicates that small RNAs (sRNAs) play an important role in disease progression as posttranscriptional regulators. The sRNA ArcZ positively regulates the motility phenotype and transcription of flagellar genes in E. amylovora Ea1189 yet is a direct repressor of translation of the flagellar master regulator, FlhD. We utilized transposon mutagenesis to conduct a forward genetic screen and identified suppressor mutations that increase motility in the Ea1189ΔarcZ mutant background. This enabled us to determine that the mechanism of transcriptional activation of the flhDC mRNA by ArcZ is mediated by the leucine-responsive regulatory protein, Lrp. We show that Lrp contributes to expression of virulence and several virulence-associated traits, including production of the exopolysaccharide amylovoran, levansucrase activity, and biofilm formation. We further show that Lrp is regulated posttranscriptionally by ArcZ through destabilization of lrp mRNA. Thus, ArcZ regulation of FlhDC directly and indirectly through Lrp forms an incoherent feed-forward loop that regulates levansucrase activity and motility as outputs. This work identifies Lrp as a novel participant in virulence regulation in E. amylovora and places it in the context of a virulence-associated regulatory network.IMPORTANCE Fire blight disease continues to plague the commercial production of apples and pears despite more than a century of research into disease epidemiology and disease control. The causative agent of fire blight, Erwinia amylovora coordinates turning on or off specific virulence-associated traits at the appropriate time during disease development. The development of novel control strategies requires an in-depth understanding of E. amylovora regulatory mechanisms, including regulatory control of virulence-associated traits. This study investigates how the small RNA ArcZ regulates motility at the transcriptional level and identifies the transcription factor Lrp as a novel participant in the regulation of several virulence-associated traits. We report that ArcZ and Lrp together affect key virulence-associated traits through integration of transcriptional and posttranscriptional mechanisms. Further understanding of the topology of virulence regulatory networks can uncover weak points that can subsequently be exploited to control E. amylovora.

Virulence-associated traits and in vitro biofilm-forming ability of Escherichia coli isolated from a major river traversing Northern India

Several strains of Escherichia coli harbor virulence traits, resulting in E. coli-related intestinal and extra-intestinal infections. Various studies have reported that extra-intestinal pathogenic E. coli (ExPEC) strains were prevalent in nonhuman reservoirs, including environmental waterways. It is therefore important to identify the pathogenic potential and/or ExPEC status of E. coli strains inhabiting the aquatic environments associated with anthropogenic activities. Besides virulence-associated genes, biofilm production also helps in the survival of E. coli in environmental waterbodies. Thus, the aim of the current study was to assess the virulence potential, ExPEC status, and biofilm-producing capability of E. coli isolated from the River Yamuna, a major river traversing the National Capital Region of Delhi, India. We also tried to discern a co-relation, if any, between virulence, biofilm formation, and antimicrobial resistance in these strains. Our results indicated that virulence-associated genes were scarce and none of the strain qualified the molecular criteria essential for ExPEC. This suggested that E. coli strains which can presumably cause human extra-intestinal infections were not prominent in the River Yamuna. However, the fact that more than 80% of the aquatic E. coli isolates were moderate and strong biofilm producers suggests that E. coli in these environments might serve as opportunistic pathogens. Also, no unequivocal association was observed between biofilm production, virulence, and β-lactamase genes in E. coli strains. As per the best of our knowledge, this is the first study where the relationship between virulence, biofilms, and antimicrobials has been examined in E. coli, isolated from an Indian urban aquatic waterbody.

Selection of Candida albicans trisomy during oropharyngeal infection results in a commensal-like phenotype.

When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.

Small alarmones (p)ppGpp regulate virulence associated traits and pathogenesis of Salmonella enterica serovar Typhi.

How Salmonella enterica serovar Typhi (S.Typhi), an important human pathogen, survives the stressful microenvironments inside the gastrointestinal tract and within macrophages remains poorly understood. We report here that S.Typhi has a bonafide stringent response (SR) system, which is mediated by (p)ppGpp and regulates multiple virulence-associated traits and the pathogenicity of the S.Typhi Ty2 strain. In an iron overload mouse model of S.Typhi infection, the (p)ppGpp0 (Ty2ΔRelAΔSpoT) strain showed minimal systemic spread and no mortality, as opposed to 100% death of the mice challenged with the isogenic wild-type strain. Ty2ΔRelAΔSpoT had markedly elongated morphology with incomplete septa formation and demonstrated severely attenuated motility and chemotaxis due to the loss of flagella. Absence of the Vi-polysaccharide capsule rendered the mutant strain highly susceptible to complement-mediated lysis. The phenotypes of Ty2ΔRelAΔSpoT was contributed by transcriptional repression of several genes, including fliC, tviA, and ftsZ, as found by reverse transcriptase quantitative polymerase chain reaction and gene complementation studies. Finally, Ty2ΔRelAΔSpoT had markedly reduced invasion into intestinal epithelial cells and significantly attenuated survival within macrophages. To the best of our knowledge, this was the first study that addressed SR in S.Typhi and showed that (p)ppGpp was essential for optimal pathogenic fitness of the organism.

The Ability of Lytic Staphylococcal Podovirus vB_SauP_phiAGO1.3 to Coexist in Equilibrium With Its Host Facilitates the Selection of Host Mutants of Attenuated Virulence but Does Not Preclude the Phage Antistaphylococcal Activity in a Nematode Infection Model.

Phage vB_SauP_phiAGO1.3 (phiAGO1.3) is a polyvalent Staphylococcus lytic podovirus with a 17.6-kb genome (Gozdek et al., 2018). It can infect most of the Staphylococcus aureus human isolates of dominant clonal complexes. We show that a major factor contributing to the wide host range of phiAGO1.3 is a lack or sparcity of target sites for certain restriction-modification systems of types I and II in its genome. Phage phiAGO1.3 requires for adsorption β-O-GlcNAcylated cell wall teichoic acid, which is also essential for the expression of methicillin resistance. Under certain conditions an exposure of S. aureus to phiAGO1.3 can lead to the establishment of a mixed population in which the bacteria and phages remain in equilibrium over multiple generations. This is reminiscent of the so called phage carrier state enabling the co-existence of phage-resistant and phage-sensitive cells supporting a continuous growth of the bacterial and phage populations. The stable co-existence of bacteria and phage favors the emergence of phage-resistant variants of the bacterium. All phiAGO1.3-resistant cells isolated from the phage-carrier-state cultures contained a mutation inactivating the two-component regulatory system ArlRS, essential for efficient expression of numerous S. aureus virulence-associated traits. Moreover, the mutants were unaffected in their susceptibility to infection with an unrelated, polyvalent S. aureus phage of the genus Kayvirus. The ability of phiAGO1.3 to establish phage-carrier-state cultures did not preclude its antistaphylococcal activity in vivo in an S. aureus nematode infection model. Taken together our results suggest that phiAGO1.3 could be suitable for the therapeutic application in humans and animals, alone or in cocktails with Kayvirus phages. It might be especially useful in the treatment of infections with the majority of methicillin-resistant S. aureus strains.

Some strains that have converged to infect Prunus spp. trees are members of distinct Pseudomonas syringae genomospecies and ecotypes as revealed by in silico genomic comparison.

A complementary taxonomic and population genetic study was performed to delineate genetically and ecologically distinct species within the Pseudomonas syringae complex by assessing 16 strains including pathovar strains that have converged to infect Prunus spp. trees, and two outgroups. Both average nucleotide identity and genome-to-genome distance comparison methods revealed the occurrence of distinct genomospecies, namely 1, 2, 3 and 8 (sensu Gardan et al.), with the latter two being closely related. Strains classified as P. s. pv. morsprunorum clustered into two distinct genomospecies, namely 2 and 8. Both the AdaptML and hierarchical Bayesian analysis of population structure methods highlighted the presence of three ecotypes, and the taxonomically related genomospecies 3 and 8 strains were members of the same ecotype. The distribution of pathogenic and virulence-associated genetic traits among Pseudomonas strains did not reveal any distinct type III secretion system effector or phytotoxin distribution pattern that characterized single genomospecies and strains that infect Prunus spp. The complete WHOP (Woody HOst and Pseudomonas spp.) genomic region and the entire β-ketoadipate gene cluster, including the catBCA operon, were found only in the members of genomospecies 2 and in the two P. s. pv. morsprunorum strains of genomospecies 8. A reduced gene flow between the three ecotypes suggested that point mutations played a larger role during the evolution of the strains than recombination. Our data support the idea that Prunus trees can be infected by different strains of distinct Pseudomonas genomospecies/ecotypes through diverse mechanisms of host colonization and infection. Such strains may represent particular lineages that emerged from environments other than that of the infected plant upon acquiring genetic traits that gave them the ability to cause plant diseases. The complementary assessment of bacterial strains using both taxonomic approaches and methods that reveal ecologically homogeneous populations has proven useful in confirming the cohesion of bacterial clusters.

Evolution of Antibiotic Resistance in Biofilm and Planktonic Pseudomonas aeruginosa Populations Exposed to Subinhibitory Levels of Ciprofloxacin.

The opportunistic Gram-negative pathogen Pseudomonas aeruginosa, known for its intrinsic and acquired antibiotic resistance, has a notorious ability to form biofilms, which often facilitate chronic infections. The evolutionary paths to antibiotic resistance have mainly been investigated in planktonic cultures and are less studied in biofilms. We experimentally evolved P. aeruginosa PAO1 colony biofilms and stationary-phase planktonic cultures for seven passages in the presence of subinhibitory levels (0.1 mg/liter) of ciprofloxacin (CIP) and performed a genotypic (whole-bacterial population sequencing) and phenotypic assessment of the populations. We observed a higher proportion of CIP resistance in the CIP-evolved biofilm populations than in planktonic populations exposed to the same drug concentrations. However, the MICs of ciprofloxacin were lower in CIP-resistant isolates selected from the biofilm population than the MICs of CIP-resistant isolates from the planktonic cultures. We found common evolutionary trajectories between the different lineages, with mutations in known CIP resistance determinants as well as growth condition-dependent adaptations. We observed a general trend toward a reduction in type IV-pilus-dependent motility (twitching) in CIP-evolved populations and a loss of virulence-associated traits in the populations evolved in the absence of antibiotic. In conclusion, our data indicate that biofilms facilitate the development of low-level mutational resistance, probably due to the lower effective drug exposure than in planktonic cultures. These results provide a framework for the selection process of resistant variants and the evolutionary mechanisms involved under the two different growth conditions.

Antimicrobial Resistance and Virulence-Associated Traits of Campylobacter jejuni Isolated From Poultry Food Chain and Humans With Diarrhea.

The objective of this study was to test the prevalence of virulence-associated markers and antimicrobial resistance in 624 C. jejuni isolated from poultry food chain, i. e., chicken feces (n = 160), poultry carcasses (n = 157), poultry meat (n = 152) and from humans (n = 155). All human strains were positive for 9 out of 13 putative virulence genes responsible for expression of pathogenic factors involved in different stages of the infection. The presence of all markers was also high in strains from chicken feces, carcasses and meat although not all of them were identified in 100% of the isolates. On the other hand, the virB11, wlaN, and iam putative pathogenic genes were detected in only 1.9, 15.2, and 20.5% of strains, respectively. C. jejuni isolates, irrespective of the origin, were highly resistant to ciprofloxacin (92.5% isolates), followed by nalidixic acid (88.9%) and tetracycline (68.4%). In case of ciprofloxacin, significantly more isolates from poultry feces, carcasses and meat were resistant than those obtained from humans and the same relationship was observed for tetracycline where the isolates from chicken feces were more often resistant than C. jejuni of carcasses and meat origin. A low number of strains was resistant to streptomycin (18.4% isolates) and only 5 strains (0.8%) displayed resistance to erythromycin. A relationship between resistance to fluoroquinolones and presence of selected pathogenic markers was observed, e.g., from 83.3% strains with the virB11 to 93.4% with the docA genes were resistant to ciprofloxacin. The isolates that did not possess any of the pathogenic traits were also mainly resistant to this antimicrobial, although the number of such strains was usually low, except virB11 (612 isolates), wlaN (529 strains), and iam (496 isolates). Furthermore, resistance to tetracycline was somehow associated with the presence of the virulence associated genes wlaN and virB11 (56.8 and 75.0% isolates, respectively). The present study shows a high antimicrobial resistance to quinolones and tetracycline of C. jejuni isolated along poultry food chain and from patients with diarrhea, which was closely correlated with the presence of several virulence genes playing a role in the pathogenesis of Campylobacter infection.

Infection by Magnaporthe oryzae chrysovirus 1 strain A triggers reduced virulence and pathogenic race conversion of its host fungus, Magnaporthe oryzae

Magnaporthe oryzae chrysovirus 1 strain A (MoCV1-A) is associated with an impaired growth phenotype of its host fungus, Magnaporthe oryzae. In this report, we assayed the virulence and pathogenicity of MoCV1-A-infected and MoCV1-A-free M. oryzae on rice plants. MoCV1-A infection did not affect virulence-associated fungal traits, such as conidial germination and appressorium formation. However, after punch inoculation of leaves on rice plants, MoCV1-A-infected strain formed smaller lesions than the MoCV1-A-free strain did on all rice varieties tested, showing that MoCV1-A infection resulted in reduced virulence of host fungi in rice plants. In contrast, after spray inoculation of rice seedlings, in some cases, MoCV1-A-infected and MoCV1-A-free strains caused different lesion types (resistance to susceptible, or vice versa) on individual international differential rice varieties. However, we did not find any gain/loss of the fungal avirulence genes by PCR, suggesting that MoCV1-A infection can convert the pathogenicity of the host M. oryzae from avirulence to virulence, or from virulence to avirulence, depending on the rice variety. We also confirmed the correlation of these race conversion events and invasive hyphae growth of the fungi in a leaf sheath inoculation assay. These data suggested that MoCV1-A infection generally confers hypovirulence to the fungal host and could be a driving force to generate physiological diversity, including pathogenic races.

Genome-Wide Screens Reveal New Gene Products That Influence Genetic Competence in Streptococcus mutans.

A network of genes and at least two peptide signaling molecules tightly control when Streptococcus mutans becomes competent to take up DNA from its environment. Widespread changes in the expression of genes occur when S. mutans is presented with competence signal peptides in vitro, including the increased production of the alternative sigma factor, ComX, which activates late competence genes. Still, the way that gene products that are regulated by competence peptides influence DNA uptake and cellular physiology are not well understood. Here, we developed and employed comprehensive transposon mutagenesis of the S. mutans genome, with a screen to identify mutants that aberrantly expressed comX, coupled with transposon sequencing (Tn-seq) to gain a more thorough understanding of the factors modulating comX expression and progression to the competent state. The screens effectively identified genes known to affect competence, e.g., comR, comS, comD, comE, cipB, clpX, rcrR, and ciaH, but disclosed an additional 20 genes that were not previously competence associated. The competence phenotypes of mutants were characterized, including by fluorescence microscopy to determine at which stage the mutants were impaired for comX activation. Among the novel genes studied were those implicated in cell division, the sensing of cell envelope stress, cell envelope biogenesis, and RNA stability. Our results provide a platform for determining the specific chemical and physical cues that are required for genetic competence in S. mutans, while highlighting the effectiveness of using Tn-seq in S. mutans to discover and study novel biological processes.IMPORTANCEStreptococcus mutans acquires DNA from its environment by becoming genetically competent, a physiologic state triggered by cell-cell communication using secreted peptides. Competence is important for acquiring novel genetic traits and has a strong influence on the expression of virulence-associated traits of S. mutans Here, we used transposon mutagenesis and genomic technologies to identify novel genes involved in competence development. In addition to identifying genes previously known to be required for comX expression, 20 additional genes were identified and characterized. The findings create opportunities to diminish the pathogenic potential of S. mutans, while validating technologies that can rapidly advance our understanding of the physiology, biology, and genetics of S. mutans and related pathogens.

Pathogenicity determinants and antibiotic resistance profiles of enterococci from foods of animal origin in Turkey.

In this study, the presence of genes responsible for the pathogenicity and antibiotic resistance profile of enterococci isolated from various foodstuffs of animal origin was investigated. The percentage prevalence of enterococci was 54.1% (203/375) and the average count was found to be 3.81 log cfu/ml-g. Species-specific primers revealed Enterococcus faecalis as the predominant species carrying one or more virulence-associated traits of efa, gelE, ace, esp and agg genetic markers. Only one E. faecium isolate (from milk) was positive for the esp gene. Regarding antibiotic resistance, the highest frequency of resistance was observed for tetracycline (21.7%), followed by quinupristin/dalfopristin (13.3%), ciprofloxacin (2.0%), penicillin (2.0%), linezolid (1.0%), ampicillin (1.0%), streptomycin (1.0%), and gentamicin (0.5%). Enterococcus faecalis showed a higher prevalence of antibiotic resistance than other enterococci. The percentage of multidrug resistance among the isolates was 3.4%. Twenty-nine E. faecalis isolates (26.6%) carrying one of the virulence-associated traits were at the same time resistant to at least one antibiotic. Our results show that foods of animal origin, including ready-to-eat products, may be reservoirs of antibiotic-resistant and potentially virulent enterococci.

Phylogenetic Backgrounds and Virulence-Associated Traits of Escherichia coli Isolates from Surface Waters and Diverse Animals in Minnesota and Wisconsin.

Possible external reservoirs for extraintestinal pathogenic Escherichia coli (ExPEC) strains that cause infections in humans are poorly defined. Because of the tremendous human health importance of ExPEC infections, we assessed surface waters and domesticated and wild animals in Minnesota and Wisconsin as potential reservoirs of ExPEC of human health relevance. We characterized 595 E. coli isolates (obtained from 1999 to 2002; 280 from seven surface water sites, 315 from feces of 13 wild and domesticated animal species) for phylogroup and virulence genotype, including inferred ExPEC status, by using multiplex PCR-based methods. We also compared the pulsed-field gel electrophoresis (PFGE) profiles of the isolates with a large private PFGE profile library. We found a predominance of non-ExPEC strains (95% and 93% among water and animal isolates, respectively), which were mainly from phylogroups A and B1, plus a minority of ExPEC strains (5% and 7% among water isolates and animal isolates, respectively), predominantly from phylogroup B2. The ExPEC strains, although significantly associated with cats, dogs, and turkeys, occurred in several additional animal species (goat, horse, chicken, pig) and were distributed broadly across all surface water sites. Virulence gene content among the animal source ExPEC isolates segregated significantly in relation to host species, following established patterns. PFGE analysis indicated that 11 study isolates closely matched (94% to 100% profile similarity) reference human clinical and fecal isolates. These findings imply what probably is a low but non-zero risk to humans from environmental and animal source E. coli isolates, especially those from specific human-associated animal species.IMPORTANCE Our detection of potentially pathogenic strains that may pose a health threat to humans among E. coli isolates from surface waters and wild and domesticated animals suggests a need for heightened attention to these reservoirs as possible sources for human acquisition of disease-causing E. coli Although cats, dogs, and turkeys were especially high-prevalence sources, the presence of such strains in other animal species and at all sampled water sites suggests that this potential risk may be widespread.

Coordinated Regulation of the EIIMan and fruRKI Operons of Streptococcus mutans by Global and Fructose-Specific Pathways.

The glucose/mannose-phosphotransferase system (PTS) permease EIIMan encoded by manLMN in the dental caries pathogen Streptococcus mutans has a dominant influence on sugar-specific, CcpA-independent catabolite repression (CR). Mutations in manL affect energy metabolism and virulence-associated traits, including biofilm formation, acid tolerance, and competence. Using promoter::reporter fusions, expression of the manLMN and the fruRKI operons, encoding a transcriptional regulator, a fructose-1-phosphate kinase and a fructose-PTS permease EIIFru, respectively, was monitored in response to carbohydrate source and in mutants lacking CcpA, FruR, and components of EIIMan Expression of genes for EIIMan and EIIFru was directly regulated by CcpA and CR, as evinced by in vivo and in vitro methods. Unexpectedly, not only was the fruRKI operon negatively regulated by FruR, but also so was manLMN Carbohydrate transport by EIIMan had a negative influence on expression of manLMN but not fruRKI In agreement with the proposed role of FruR in regulating these PTS operons, loss of fruR or fruK substantially altered growth on a number of carbohydrates, including fructose. RNA deep sequencing revealed profound changes in gene regulation caused by deletion of fruK or fruR Collectively, these findings demonstrate intimate interconnection of the regulation of two major PTS permeases in S. mutans and reveal novel and important contributions of fructose metabolism to global regulation of gene expression.IMPORTANCE The ability of Streptococcus mutans and other streptococcal pathogens to survive and cause human diseases is directly dependent upon their capacity to metabolize a variety of carbohydrates, including glucose and fructose. Our research reveals that metabolism of fructose has broad influences on the regulation of utilization of glucose and other sugars, and mutants with changes in certain genes involved in fructose metabolism display profoundly different abilities to grow and express virulence-related traits. Mutants lacking the FruR regulator or a particular phosphofructokinase, FruK, display changes in expression of a large number of genes encoding transcriptional regulators, enzymes required for energy metabolism, biofilm development, biosynthetic and degradative processes, and tolerance of a spectrum of environmental stressors. Since fructose is a major component of the modern human diet, the results have substantial significance in the context of oral health and the development of dental caries.

Characterization of (Uropathogenic) E. coli isolated from urinary tract infections: phylogenetic typing and distribution of virulence-associated traits

Uropathogenic Escherichia coli (UPEC) is the pre-dominant bacterial pathogen causing Urinary Tract infections (UTIs). Pathogenicity of UPEC results from presence and expression of several virulence...

Pseudomonas aeruginosa quorum-sensing response in the absence of functional LasR and LasI proteins: the case of strain 148, a virulent dolphin isolate.

Pseudomonas aeruginosa is an opportunistic pathogen that presents a complex regulatory network called 'quorum-sensing', which is responsible for the transcription of genes coding for several traits implicated in its pathogenicity. Strain 148 is a dolphin isolate that has been shown to produce quorum-sensing-regulated virulence traits and to be virulent in a mouse model, despite the fact that it contains a 20-kbp deletion that eliminates from the chromosome the lasR gene and the lasI promoter. LasR is a key quorum-sensing transcriptional regulator that, when coupled with the autoinducer 3-oxo-dodecanoyl homoserine lactone (3O-C12-HSL) produced by LasI, activates transcription of genes coding for some virulence-associated traits such as elastase, lasI, rhlI and rhlR. RhlR is also a key quorum-sensing transcriptional regulator that, when interacting with the autoinducer butanoyl homoserine lactone (C4-HSL) that is produced by the synthase RhlI, activates the genes involved in the synthesis of some virulence-associated traits, as rhamnolipids and pyocyanin. We describe that in P. aeruginosa 148, the LasR/3O-C12-HSL-independent rhlR transcriptional activation is due to the release of the negative effect of Vfr (a CRP-ortholog) caused by the insertion of an IS element in vfr, and that rhlI transcription is driven from the rhlR promoter, forming the rhlR-I operon.

The Snf1-activating kinase Sak1 is a key regulator of metabolic adaptation and in vivo fitness of Candida albicans.

The metabolic flexibility of the opportunistic fungal pathogen Candida albicans is important for colonisation and infection of different host niches. Complex regulatory networks, in which protein kinases play central roles, link metabolism and other virulence-associated traits, such as filamentous growth and stress resistance, and thereby control commensalism and pathogenicity. By screening a protein kinase deletion mutant library that was generated in the present work using an improved SAT1 flipper cassette, we found that the previously uncharacterised kinase Sak1 is a key upstream activator of the protein kinase Snf1, a highly conserved regulator of nutrient stress responses that is essential for viability in C. albicans. The sak1Δ mutants failed to grow on many alternative carbon sources and were hypersensitive to cell wall/membrane stress. These phenotypes were mirrored in mutants lacking other subunits of the SNF1 complex and partially compensated by a hyperactive form of Snf1. Transcriptional profiling of sak1Δ mutants showed that Sak1 ensures basal expression of glyoxylate cycle and gluconeogenesis genes even in glucose-rich media and thereby contributes to the metabolic plasticity of C. albicans. In a mouse model of gastrointestinal colonisation, sak1Δ mutants were rapidly outcompeted by wild-type cells, demonstrating that Sak1 is essential for the in vivo fitness of C. albicans.

Capsular Polysaccharide Types and Virulence-Related Traits of Epidemic KPC-Producing Klebsiella pneumoniae Isolates in a Chinese University Hospital.

Klebsiella pneumoniae is an important human pathogen associated with a variety of diseases and the prevalence of blaKPC carrying K. pneumoniae (KPC-Kp) is rapidly increasing. Capsule is an important virulence factor in K. pneumoniae. In this study, we determined to first systematically characterize capsular polysaccharide (CPS) and virulence traits in KPC-Kp strains. A total of 56 KPC-Kp isolates were recovered from clinical samples in a Chinese hospital, which were assigned to clonal lineages by multilocus sequence typing (MLST). Capsule typing (wzi sequencing and wzc polymerase chain reaction [PCR]) and virulence genes were characterized by molecular approaches. The virulence of these strains was determined by biofilm formation, serum killing resistance, phagocytosis, and infection models. Six different STs were found among 56 KPC-Kp isolates: 76.8% (43 of 56 isolates) belonged to ST11, 6 isolates belonged to ST147, 4 isolates belonged to ST15, 1 isolate belonged to ST1456, 1 isolate belonged to ST65, and 1 isolate was ST23. Based on the wzi gene DNA sequences and wzc PCR, these 56 strains were classified as capsular type wzi47-K47 (n = 37), wzi64-K64 (n = 8), wzi8-K8 (n = 4), wzi37-K37 (n = 4), wzi53-K53 (n = 1), wzi125-K2 (n = 1), and wzi1-K1 (n = 1). Heterogeneity was detected in biofilm formation and phagocytosis among different CPS types. ST11 strains were less virulent than other ST strains. KPC-Kp strains exhibit variability of virulence-associated traits. Differences were associated with the ST types and CPS.

Role of ppGpp in Pseudomonas aeruginosa acute pulmonary infection and virulence regulation

During infection, bacteria might generate adaptive responses to facilitate their survival and colonization in the host environment. The alarmone guanosine 5′-triphosphate-3′-diphosphate (ppGpp), the levels of which are regulated by the RelA and SpoT enzymes, plays a critical role in mediating bacterial adaptive responses and virulence. However, the mechanism by which ppGpp regulates virulence-associated traits in Pseudomonas aeruginosa is poorly understood. To investigate the regulatory role of ppGpp, the ppGpp-deficient strain ΔRS (relA and spoT gene double mutant) and the complemented strain ΔRS++ (complemented with relA and spoT genes) were constructed. Herein, we reported that the ΔRS strain showed decreased cytotoxicity towards A549 human alveolar adenocarcinoma cell lines and led to reduced mortality, lung edema and inflammatory cell infiltration in a mouse model of acute pneumonia compared to wild-type PAO1 and the complemented strain ΔRS++. Subsequent analyses demonstrated that the ΔRS strain displayed reduced T3SS expression, decreased levels of elastase activity, pyocyanin, pyoverdin and alginate, and inhibited swarming and biofilm formation compared to PAO1 and the complemented strain ΔRS++. In addition, the results demonstrate that ppGpp-mediated regulation of T3SS, virulence factor production, and swarming occurs in a quinolone quorum-sensing system-dependent manner. Taken together, these results suggest that ppGpp is required for virulence regulation in P. aeruginosa, providing new clues for the development of interference strategies against bacterial infection.