Relative Virulence Research Articles

Dual RNA-seq of Orientia tsutsugamushi informs on host-pathogen interactions for this neglected intracellular human pathogen

Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.

Indole signaling decreases biofilm formation and related virulence of Listeria monocytogenes

Bacterial communication system known as quorum sensing (QS) is a pivotal system for bacterial survival, adaptation and pathogenesis. Members in the multicellular community may synthesize or acquire a signaling molecule in order to elicit downstream cellular processes. Roles of indole and derivatives, a new class of quorum-sensing signal molecules, in various bacterial physiologies and virulence have been reported recently. Indole is normally found in mammal gastrointestinal tract as a metabolite of tryptophan metabolism by microbiota. Therefore, interspecies connection via indole signaling among commensal bacteria and enteric pathogens could be anticipated. Effects of indole exposure on the virulence of Listeria monocytogenes were investigated by phenotypic and molecular approaches. Results demonstrated that synthetic indole and indole-rich conditioned medium significantly diminished biofilm formation and related virulence of L. monocytogenes including motility, cell aggregation and exopolysaccharide production. Transcript levels of virulence-associated (pssE, dltA, flaA, fliI, motB, agrA and hly) and regulatory genes (codY, sigB, prfA and gmaR) were substantially downregulated in indole-treated cells. Only mogR gene encoding for a repressor of motility genes was upregulated after indole exposure. Our findings raise the possibility that L. monocytogenes may acquire indole signaling from gut microbiota for resource-effective adaptation upon transition to new environment.

Stachybotriaceae on Cucurbits Demystified: Genetic Diversity and Pathogenicity of Ink Spot Pathogens.

Recently, the incidence of Myrothecium leaf spot, a foliar disease of watermelon, has increased in South Carolina. However, the identity of the fungal species responsible for outbreaks of this disease has not been determined. Sequence data from four partial gene regions were used to conduct Bayesian inference in order to identify 95 isolates of Stachybotriaceae. Isolates were collected in South Carolina between July 2015 and May 2018. In total, six species of Stachybotriaceae were identified on watermelon and two other cucurbits: Albifimbria verrucaria, Gregatothecium humicola, Paramyrothecium foliicola, P. humicola, Xenomyrothecium tongaense, and Xepicula leucotricha. Two species, G. humicola and P. foliicola, were the predominant species found. Within these two species, genetic differences within small spatial scales were detected. Five species (all except Xenomyrothecium tongaense) were tested in experiments to determine their pathogenicity and relative virulence on three hosts grown in rotation in South Carolina. Southern pea plants were less susceptible than watermelon and tomato plants, which were equally susceptible. This constitutes the first reliable report of pathogenicity of any of the five tested species of Stachybotriaceae on these three vegetable crops. Another important finding was that none of the isolates were identified as P. roridum, the species considered to be the only causal agent of Myrothecium leaf spot on cucurbits. We propose the common name "ink spot" for the foliar phase of diseases caused by genera within the family Stachybotriaceae. This name is descriptive and likely to be accepted by growers. To prevent further loss incurred by ink spot, watermelon and tomato crops should be monitored for this disease.

Species Variety, Antibiotic Susceptibility Patterns and Prevalence of Enterotoxin Genes in Staphylococci Isolated from Foodstuff in Central Iran

The presence and diversity of Staphylococcus species and their enterotoxin-encoding genes in foodstuffs have not been comprehensively studied in some developing countries. This study aimed to assess the frequency of Staphylococcus spp. and their related virulence factors in foodstuffs in Isfahan, Iran. Overall, 139 foodstuff samples, collected from Isfahan City (center of Iran) from Sep 2015 to Oct 2016, were processed for the presence of Staphylococcus spp. using standard bacteriological procedures and sequence analysis of 16S rRNA gene. Antimicrobial susceptibilities and prevalence of mecA and toxin-encoded genes (sea, seb, sed, see and tsst1 ) were tested for all of the Staphylococcal isolates. Forty-four Gram-positive cocci were recovered from 139 dairy and meat samples. The most prevalent species were S. vitulinus 25.0% (11/44) and S. aureus 20.5% (9/44); respectively. The most prevalent antimicrobial resistance was noted towards penicillin, cefoxitin and tetracycline. The sec, sea, see and tsst1 genes were found in 19%, 9.5%, 3.5%, and 3.5% of the isolates, respectively. Numerous virulence factors were detected in different Staphylococcus spp. isolated from foodstuffs, more attention should be paid to the presence of the bacteria. Proper hygienic and management practices should be considered in order to increase food safety and prevent extra treatment costs.

Inhibitory activity of metal-curcumin complexes on quorum sensing related virulence factors of Pseudomonas aeruginosa PAO1

The use of metal complexes to reduce or inhibit virulence factors of Pseudomonas aeruginosa is a promising strategy for the management and control of infections caused by this multidrug-resistant pathogen. The present study aimed to investigate the anti-quorum sensing activity of sub-minimum inhibitory concentrations (sub-MIC) of copper(II) sulfate pentahydrate-curcumin complex (Cu-CUR), iron(III) nitrate nonahydrate -curcumin complex (Fe-CUR), zinc(II) chloride-curcumin complex (Zn-CUR) and free curcumin (free-CUR) against P. aeruginosa PAO1. Metal-CUR complexes were synthesized and characterized by spectroscopic methods. The effect of sub-MIC (1/4 and 1/16 MIC) concentrations of metal-CUR complexes and free-CUR on cell growth, biofilm formation, motility, alginate and pyocyanin production, H2O2 susceptibility and expression of lasI and lasR genes in PAO1 was determined. MIC of metal-CUR complexes and free-CUR was determined as 62.5 and 125 µg/ml, respectively. Metal-CUR complexes at concentration of 62.5 µg/ml significantly reduced the cell growth to 1.5%–3.3%. Although we did not measure the anti-QS activity of metal-CUR complexes directly against PAO1, they indicated anti-QS activity in C. violaceum CV026. Copper-CUR complex at the concentration of 1/4 MIC showed the greatest inhibitory effect on swarming and twitching motilities, biofilm formation, alginate and pyocyanin production, sensitivity to H2O2 and reduction in the expression levels of lasI and lasR genes (P < 0.001). Considering the biological effects of Cu-CUR complex and its inhibitory activity on virulence factors, it may be used as an effective compound for treatment and control of infections caused by P. aeruginosa.

Differential immune cell response to Mycobacterium intracellulare colony morphotypes

Abstract Mycobacterium intracellulare is an opportunistic pathogen which causes disease in immunocompromised individuals. Like several other nontuberculous mycobacteria (NTM) species, a single strain of M. intracellulare forms bacterial colonies with varying morphologies; however, the relative virulence and immunogenicity of differing M. intracellulare morphotypes are unknown. We have obtained a strain of M. intracellulare isolated from an infected patient and further subcultured it to distinguish the component colony morphotypes present within the parent isolate. To determine whether these distinct colony morphotypes have the ability to stimulate differential immune responses in an infected host, C57BL/6J mice were aerosol infected with either the parent clinical strain or each of the isolated component colony morphotypes. Differential immune response and virulence characteristics of the morphotypes were evaluated by tracking host bacterial load, cytokine expression profiles, and histology. In addition, bone marrow-derived macrophages were isolated and infected in vitro with the different morphotypes and parent isolate. From this study, we have found that the component colony morphotypes are able to differentially activate macrophages and affect the host immune response to infection. These results may establish an important immunological basis for patient clinical outcomes.

Prevalence of Escherichia coli K1 Rectovaginal Colonization Among Pregnant Women in Iran: Virulence Factors and Antibiotic Resistance Properties.

Neonatal invasive infections caused by Escherichia coli K1 are still major health problems and effective preventive strategies at the maternal level can be a concern. The aim of this study was to determine the prevalence of rectovaginal colonization, related risk factors, virulence factors, and antibiotic resistance properties of E. coli K1 among pregnant women. In this cross-sectional study, vaginal and rectal swabs were collected from 400 pregnant women. The identification of E. coli isolates was performed by microbiological tests. A polymerase chain reaction (PCR) assay was used to identify the E. coli K1 strains. The antimicrobial susceptibility patterns were determined by the Kirby-Bauer disk diffusion. Two duplex PCR assays were developed separately to detect genes encoding virulence determinants (fimH, hlyF, ibeA, and iucC) in the E. coli strains. The vaginal and rectal maternal E. coli K1 colonization rates were 3.7% and 19.25%, respectively. There is no significant association between demographic-obstetric factors and vaginal E. coli colonization in pregnant women. The most effective antibiotics against E. coli K1 strains were imipenem, gentamycin, ciprofloxacin, and ceftazidime. In our study, the E. coli K1 strains were significantly more likely to possess the fimH (90.9% vs. 60.7%) and iucC (90.9% vs. 53.6%) than the E. coli non-K1 strains. This study demonstrates that E. coli K1 seems to be more virulent than non-K1 strains. Our findings highlight the importance of screening pregnant women for vaginal colonization by E. coli K1 and of the appropriate antibiotic prophylaxis for the prevention of early-onset E. coli neonatal infection and comorbidity.

Inhibition of the pqsABCDE and pqsH in the pqs quorum sensing system and related virulence factors of the Pseudomonas aeruginosa PAO1 strain by farnesol

Farnesol, a 15-carbon, naturally occurring sesquiterpene alcohol, exists widely in plants and Candida albicans. It inhibits the production of the Pseudomonas Quinolone Signal (PQS) and pyocyanin of the opportunistic pathogen Pseudomonas aeruginosa, but the underlying mechanisms remained unknown. In this study, the actions of farnesol on the growth, production of virulence factors (including PQS signals, elastase, pyocyanin, biofilm, and swarming motility), and the transcription of some essential key genes of three Quorum Sensing (QS) systems and other related virulence genes in the P. aeruginosa PAO1 strain were investigated. Moreover, the qRT-PCR and high throughput transcriptome/proteome sequencing techniques were used to differentiate the transcriptomic/proteomic expressions of the PAO1 strain following farnesol exposure. The experimental results showed that farnesol did not inhibit PAO1 growth or affect its elastase production. However, farnesol did suppress the transcription and protein expression of the pqsABCDE and pqsH genes in the strain's pqs QS system and then inhibit the PQS production, while leaving the transcription and protein expression of pqsR in the pqs system, of lasI and lasR in the las QS system, and of rhlI and rhlR in the rhl QS system unaffected. Farnesol also inhibited the expression of some related virulence genes and the production of the related virulence factors. For example, it inhibited the protein expression of key genes in the phenazine biosynthesis pathway, including key genes related to pyocyanin synthesis (phzM, phzS), inhibiting the strain's capacity for biofilm formation. In addition, farnesol enhanced the transcription and protein expression of key genes in the geraniol degradation pathway. Farnesol also stimulated the PAO1 strain with regard to swarming motility. As a result, we concluded that farnesol can suppress the virulence by inactivating the pqs QS system in pathogen P. aeruginosa, which would be significant to develop a kind of effective drug control the virulence and pathogenesis of this bacteria.

Synthesis, characterization and\xa0assessment of anti-quorum sensing activity of copper(II)-ciprofloxacin complex against Pseudomonas aeruginosa PAO1

Quorum sensing (QS) inhibition by metal-antibiotic complexes is a promising strategy for the management and control of multidrug resistant Pseudomonas aeruginosa infections. We investigated the anti-quorum sensing activity of sub-minimum inhibitory concentration (sub-MIC) of copper(II) sulfate pentahydrate-ciprofloxacin (Cu-CIP) complex and free ciprofloxacin (free-CIP) against P. aeruginosa PAO1. Copper-CIP complex was synthesized and its characterization was assessed using spectroscopic methods and single crystal X-ray analysis. The effect of sub-MIC (1/4 and 1/16 MIC) concentrations of Cu-CIP and free-CIP on cell growth, biofilm formation, motility, alginate and pyocyanin production, H2O2 susceptibility and expression of QS circuit genes lasI and lasR in PAO1 was determined. Minimum inhibitory concentration of Cu-CIP complex and free-CIP was determined as 0.125 µg/ml. Copper-CIP complex did not show significant effect on the cell growth at concentrations of 1/4 and 1/16 MIC. However, sub-MIC concentrations (1/4 and 1/16 MIC) of Cu-CIP showed the significant reduction in violacein production, motility, biofilm formation, alginate and pyocyanin production and sensitivity to H2O2 in a concentration dependent manner (P < 0.001). Copper-CIP at the concentration of 1/4 MIC showed the greatest reduction in lasI and lasR transcriptional expression (89.5% and 96.2% respectively). Considering the biological effects of Cu-CIP complex and its inhibitory activity on QS related virulence traits at low concentrations (0.03 and 0.007 µg/ml), it may be used as an effective approach in the management of infections caused by P. aeruginosa.

Acquisition of plasmids conferring carbapenem and aminoglycoside resistance and loss of surface-exposed macromolecule structures as strategies for the adaptation of Acinetobacter baumannii CC104O/CC15P strains to the clinical setting.

Acinetobacter baumannii (Aba) is an emerging opportunistic pathogen associated to nosocomial infections. The rapid increase in multidrug resistance (MDR) among Aba strains underscores the urgency of understanding how this pathogen evolves in the clinical environment. We conducted here a whole-genome sequence comparative analysis of three phylogenetically and epidemiologically related MDR Aba strains from Argentinean hospitals, assigned to the CC104O/CC15P clonal complex. While the Ab244 strain was carbapenem-susceptible, Ab242 and Ab825, isolated after the introduction of carbapenem therapy, displayed resistance to these last resource β-lactams. We found a high chromosomal synteny among the three strains, but significant differences at their accessory genomes. Most importantly, carbapenem resistance in Ab242 and Ab825 was attributed to the acquisition of a Rep_3 family plasmid carrying a bla OXA-58 gene. Other differences involved a genomic island carrying resistance to toxic compounds and a Tn10 element exclusive to Ab244 and Ab825, respectively. Also remarkably, 44 insertion sequences (ISs) were uncovered in Ab825, in contrast with the 14 and 11 detected in Ab242 and Ab244, respectively. Moreover, Ab825 showed a higher killing capacity as compared to the other two strains in the Galleria mellonella infection model. A search for virulence and persistence determinants indicated the loss or IS-mediated interruption of genes encoding many surface-exposed macromolecules in Ab825, suggesting that these events are responsible for its higher relative virulence. The comparative genomic analyses of the CC104O/CC15P strains conducted here revealed the contribution of acquired mobile genetic elements such as ISs and plasmids to the adaptation of A. baumannii to the clinical setting.

White spot syndrome virus in decapods from Mississippi Sound, USA, and susceptibility of Palaemonetes pugio and Uca panacea to a Chinese isolate.

The presence and quantity of white spot syndrome virus (WSSV) was surveyed using TaqMan real-time PCR to assess the extent of the virus in Mississippi Sound, USA. A total of 3577 wild decapods comprising 11 species was collected between November 2012 and August 2015: WSSV was present in 10 of the 11 species. Prevalence ranged from 5.1% in Uca rapax to 38.8% in U. spinicarpa. Viral load ranged from 1.8 to 7.3 log10 copies of WSSV µg-1 total DNA. Two Gulf species, Palaemonetes pugio and U. panacea, were injected with a series of doses of a virulent WSSV isolate from China to determine relative susceptibility and virulence because continuing translocation of highly pathogenic isolates of WSSV poses risk to native species. Survival was 0-65% for P. pugio and 5-60% for U. panacea. Median survival time was lower for P. pugio than U. panacea at all doses. Mean (±SD) lethal load was 9.0 ± 8.9 log copies of WSSV µg-1 total DNA in P. pugio and 8.2 ± 8.3 in U. panacea. Mean viral load in survivors was higher in U. panacea than in P. pugio (5.8 ± 6.1 vs. 3.2 ± 3.0 log copies of WSSV µg-1 total DNA); mean viral load was lower in wild individuals of those species (2.9 ± 3.2 for P. pugio and 4.9 ± 5.0 for U. panacea). U. panacea is potentially more tolerant of WSSV than P. pugio and may serve as an important reservoir host in the community.

First comparison of French and Australian OsHV-1 µvars by bath exposure.

Economically devastating mortality events of farmed and wild shellfish due to infectious disease have been reported globally. Currently, one of the most significant disease threats to Pacific oyster Crassostrea gigas culture is the ostreid herpesvirus 1 (OsHV-1), in particular the emerging OsHV-1 microvariant genotypes. OsHV-1 microvariants (OsHV-1 µvars) are spreading globally, and concern is high among growers in areas unaffected by OsHV-1. No study to date has compared the relative virulence among variants. We provide the first challenge study comparing survival of naïve juvenile Pacific oysters exposed to OsHV-1 µvars from Australia (AUS µvar) and France (FRA µvar). Oysters challenged with OsHV-1 µvars had low survival (2.5% exposed to AUS µvar and 10% to FRA µvar), and high viral copy number as compared to control oysters (100% survival and no virus detected). As our study was conducted in a quarantine facility located ~320 km from the ocean, we also compared the virulence of OsHV-1 µvars using artificial seawater made from either facility tap water (3782 µmol kg-1 seawater total alkalinity) or purchased distilled water (2003 µmol kg-1). Although no differences in survival or viral copy number were detected in oysters exposed to seawater made using tap or distilled water, more OsHV-1 was detected in tanks containing the lower-alkalinity seawater, indicating that water quality may be important for virus transmission, as it may influence the duration of viral viability outside of the host.

Two Prevalent Listeria ivanovii subsp. ivanovii Clonal Strains With Different Virulence Exist in Wild Rodents and Pikas of China.

Listeria ivanovii subsp. ivanovii is an intracellular bacterium distributed widely in nature, causing the listeriosis in ruminants and humans. Previous researches had isolated 116 strains of L. ivanovii subsp. ivanovii from wild rodents and pikas of different regions in China, and the predominant sequence types were ST1 and ST2. In this study, we first investigated the biological characteristics and virulence of these two clonal strains including motility, metabolism and virulence in cells and mouse model. The results demonstrated the ST1 strains exhibited motility, wide metabolic activity and hypervirulence, whereas the ST2 strains showed non-motility, relative lower metabolic activity and virulence. Considering the transmissible ability from wild rodents and pikas to ecological environment, the L. ivanovii subsp. ivanovii with potential pathogenicity to humans and ruminants should be monitored.

Surface Antigen 1 Is a Crucial Secreted Protein That Mediates Babesia microti Invasion Into Host Cells.

Babesia microti, a tick-borne intraerythrocytic zoonotic protozoan, causes most of human babesiosis in the world, and patients usually experience intermittent fever, fatigue, and chills, followed by a combination of additional symptoms and even death in severe cases. Unfortunately, there is no curable drug or effective vaccine available, and the mechanism of related virulence factors in invasion to host cells during the merozoite stage is unclear. Here, we evaluated a secreted protein annotated as B. microti surface antigen 1 (BmSA1) and identified from in vitro culture supernatant by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). BmSA1 fragment was expressed in Escherichia coli to prepare polyclonal antiserum. Western blot analysis revealed the existence of BmSA1 in the lysate of the parasites and the hemolysate of infected red blood cells (iRBCs). Laser confocal microscopy confirmed BmSA1 as a secreted protein with diffuse distribution around the parasites in red blood cells (RBCs). The adhesion capacity of BmSA1 against the host RBCs was tested by RBC binding assays using the recombinant BmSA1 protein (rBmSA1), which was shown to specifically bind to host RBCs. Further in vitro antiserum-neutralization test demonstrated that the growth of parasites could be significantly inhibited by the anti-BmSA1 antiserum. These results indicate that BmSA1 is a crucial factor for B. microti invasion into host RBCs with an important role in host-parasite interactions during the merozoite stage and has the potential use as a vaccine candidate due to its high secretion amount.

Proteogenomic analysis of the Clostridium difficile exoproteome reveals a correlation between phylogenetic distribution and virulence potential

C. difficile induces antibiotic-associated diarrhea due to the action of two secreted toxins, TcdA and TcdB. A considerable range of virulence among C. difficile strains has been widely reported. During a hospital outbreak, 46 isolates were collected that belonged to different genotypes. Of those, the majority corresponded to two virulent strains, the globally distributed Sequence Type 1 (ST1)_North American Pulsotype 1 (NAP1) and the endemic ST54_NAPCR1 genotypes, respectively. Whereas the virulence of the latter has been attributed to increased secretion of toxins and production of a highly cytotoxic TcdB, these characteristics do not explain the increased lethality of the former. We undertook a proteomic comparative approach of the isolates participating in the outbreak to look for proteins present in the exoproteome of the ST1_NAP1and ST54_NAPCR1 strains. We used a low virulent ST2_NAP4 strain isolated also in the outbreak as control. Dendrograms constructed using the exoproteomes of the strains were very similar to those created using genomic information, suggesting an association between secreted proteins and relative virulence of the strains. By 2D electrophoresis and mass spectrometry it was found that approximately half of the proteins are shared among strains of different genotypes. From the identified proteins, the surface-located SlpA draw our attention due to its detection in ST54_NAPCR1 exoproteomes. Biochemical analysis indicated that the processing of SlpA is different in the ST54_NAPCR1 strain and confirmed that this strain secretes more SlpA than its counterparts. Furthermore, SlpA from the ST54_NAPCR1 strain exerted an increased proinflammatory activity. Altogether, these results indicate that the exoproteome composition correlates with the C. difficile genotype and suggest that particular proteins secreted by some strains could synergize with the effects of TcdA and TcdB increasing their virulence.

In vitro and In vivo Fitness of Clinical Isolates of Carbapenem-Resistant and -Susceptible Acinetobacter baumannii

AbstractContext:Acinetobacter baumannii is one among the leading nosocomial pathogens in the healthcare settings worldwide. Limited data on relative fitness and virulence of carbapenem-resistant A. baumannii (CRAB) are known. New methods are required to curb the rapidly rising antimicrobial resistance of this bug. Aims: We aimed to study the comparative in vitro and in vivo fitness of clinical isolates of CRAB and carbapenem-susceptible A. baumannii (CSAB). Settings and Design: A total of nine A. baumannii isolates were included in this study. CSAB ATCC-19606 was taken as a reference control strain. Subjects and Methods: Matrix-assisted laser desorption ionisation–time of flight mass spectrometry and gyrB and blaOXA-51 PCR were used for species identification. Antimicrobial susceptibility was performed using Kirby-Bauer disk-diffusion method. Minimum inhibitory concentration for carbapenems (imipenem, meropenem and doripenem) was determined using agar dilution method. End point analysis, competitive index (CI), growth kinetics and generation time were determined for CRAB and CSAB isolates. In vivo fitness of CRAB and CSAB was determined using Caenorhabditis elegans host model. Multilocus sequence typing was performed to see the genetic relatedness of the isolates under study. Results: End point analysis, in vitro CI and growth kinetics experiments showed better fitness of clinical isolates of CRAB over CSAB ones. In vivo ‘nematode fertility assay’ using C. elegans also supported the in vitro results. Conclusions: To the best of our knowledge, this is the first study of its kind from India showing difference in fitness of clinical isolates of CRAB and CSAB.

Quorum quenching activity of Aerva lanata against Catheter Associated Urinary Tract Infections (CAUTI) Pathogens

Background. CAUTI is the most common hospital infection caused by Gram-negative bacterium owing to their ability to colonize UT mucosa. These uro-pathogens elaborate quorum sensing (QS) molecules that modify the expression and secretion of biofilm matrix compounds in response to rheological and other UT changes during catheterization. Quenching the biological effects of QS molecules will have substantial clinical value. This study identifies and evaluate the antibiofilm/anti-virulence properties of Aerva lanata plant extract against select CAUTI pathogens. Methods. Methanolic extract of Aerva lanata was screened for its anti-urobacterial effects using Chromobacterium violaceum as the test strain. A. lanata extract impact on the QS molecule, acyl homoserine lactone (AHL) regulated bacterial functions essential for biofilm formation was investigated. It includes RT-PCR analysis of QS genes in the bacterium and in silico studies of bacterial compounds. Results. Methanolic extract of A. lanata interfered with AHL regulated physiological functions such as biofilm formation, pellicle inhibition, flagellar motility and exopolysaccharides (EPS) production. In situ-visualization of biofilm with confocal microscopy showed concentration influenced reduction in bacterial biofilm formation in response to plant extract. GC-MS identified 11 compounds and molecular docking analysis predicted their respective host receptors. In silico analysis of plant extract revealed putative compounds with potential to inhibit the bacterial QS AHL system. RT-PCR analysis revealed down regulation of QS related virulence genes viz., las1, lasR, lasB, lasA. Conclusion. Data obtained in this study strongly suggests that Aerva lanata should be further investigated for its therapeutic potentials to treat bacterial CAUTI.

Characterization of an intermolecular quaternary interaction between discrete segments of the Streptococcusmutans adhesin P1 by NMR spectroscopy.

Cell surface-localized P1 adhesin (aka Antigen I/II or PAc) of the cariogenic bacterium Streptococcusmutans mediates sucrose-independent adhesion to tooth surfaces. Previous studies showed that P1's C-terminal segment (C123, AgII) is also liberated as a separate polypeptide, contributes to cellular adhesion, interacts specifically with intact P1 on the cell surface, and forms amyloid fibrils. Identifying how C123 specifically interacts with P1 at the atomic level is essential for understanding related virulence properties of S.mutans. However, with sizes of ~51 and ~185kDa, respectively, C123 and full-length P1 are too large to achieve high-resolution data for full structural analysis by NMR. Here, we report on biologically relevant interactions of the individual C3 domain with A3VP1, a polypeptide that represents the apical head of P1 as it is projected on the cell surface. Also evaluated are C3's interaction with C12 and the adhesion-inhibiting monoclonal antibody (MAb) 6-8C. NMR titration experiments with 15 N-enriched C3 demonstrate its specific binding to A3VP1. Based on resolved C3 assignments, two binding sites, proximal and distal, are identified. Complementary NMR titration of A3VP1 with a C3/C12 complex suggests that binding of A3VP1 occurs on the distal C3 binding site, while the proximal site is occupied by C12. The MAb 6-8C binding interface to C3 overlaps with that of A3VP1 at the distal site. Together, these results identify a specific C3-A3VP1 interaction that serves as a foundation for understanding the interaction of C123 with P1 on the bacterial surface and the related biological processes that stem from this interaction. DATABASE: BMRB submission code: 27935.

Circulating concentration of interleukin-37 in Helicobacter pylori-infected patients with peptic ulcer: Its association with IL-37 related gene polymorphisms and bacterial virulence factor CagA

The immunopathologic responses play a major role in the development of H. pylori (HP)-related gastrointestinal diseases. IL-37 is an anti-inflammatory cytokine with potent suppressive effects on innate and adaptive immune responses. Here, we investigated the IL-37 levels and two single nucleotide polymorphisms (SNPs) including rs3811047 and rs2723176 in IL-37 gene in HP-infected peptic ulcer (PU) patients to identify any relationship. Three groups, including 100 HP-infected PU patients, 100 HP-infected asymptomatic (AS) subjects and 100 non-infected healthy control (NHC) subjects were enrolled to study. Serum IL-37 levels and the genotyping at rs3811047 and rs2723176 were determined using ELISA and SSP–PCR methods, respectively. Significantly higher IL-37 levels were observed in PU patients compared with AS and NHC groups (P < 0.0001). In both PU and AS groups, the CagA+ HP-infected participants displayed higher IL-37 levels compared with those infected with CagA− strains (P < 0.0001). There were significant differences between PU, AS and NHC groups regarding the distribution of genotypes and alleles at rs3811047 and rs2723176 SNPs. The genotype GG and allele G at IL-37 rs3811047 SNP, and the genotype CC and allele C at IL-37 rs2723176 SNP more frequently expressed in PU patients than total healthy subjects (AS + NHC groups) and were associated with an increased risk of PU development (genotype GG: RR = 3.08, P < 0.009; allele G: RR = 2.94, P < 0.01; genotype CC: RR = 5, P < 0.01; and allele C: RR = 5.0, P < 0.02, respectively). The PU patients with allele A at IL-37 rs2723176 SNP expressed higher amounts of IL-37 compared with patients carried allele C at the same position (P < 0.05). In AS carriers and NHC individuals, the IL-37 levels in subjects carried genotype AA or allele A at IL-37 rs2723176 SNP were higher than those carried genotype CC or allele C at the same location (P < 0.01 and P < 0.02 for AS group; P < 0.0001 and P < 0.001 for NHC subjects, respectively). The increased IL-37 levels may be considered as a valuable marker of PU development in HP-infected individuals. The SNPs rs3811047 and rs2723176 were associated with PU development. The CagA status of HP and IL-37 rs2723176 SNP may affect the IL-37 levels.

Exploiting Correlations between Protein Abundance and the Functional Status of saeRS and sarA To Identify Virulence Factors of Potential Importance in the Pathogenesis of Staphylococcus aureus Osteomyelitis.

We used a murine model of postsurgical osteomyelitis (OM) to evaluate the relative virulence of the Staphylococcus aureus strain LAC and five isogenic variants that differ in the functional status of saeRS and sarA relative to each other. LAC and a variant in which saeRS activity is increased (saeC) were comparably virulent to each other, while ΔsaeRS, ΔsarA, ΔsaeRS/ΔsarA, and saeC/ΔsarA mutants were all attenuated to a comparable degree. Phenotypic comparisons including a mass-based proteomics approach that allowed us to assess the number and abundance of full-length proteins suggested that mutation of saeRS attenuates virulence in our OM model owing primarily to the decreased production of S. aureus virulence factors, while mutation of sarA does so owing to protease-mediated degradation of these same virulence factors. This was confirmed by demonstrating that eliminating protease production restored virulence to a greater extent in a LAC sarA mutant than in the isogenic saeRS mutant. Irrespective of the mechanism involved, mutation of saeRS or sarA was shown to result in reduced accumulation of virulence factors of potential importance. Thus, using our proteomics approach we correlated the abundance of specific proteins with virulence in these six strains and identified 14 proteins that were present in a significantly increased amount (log2 ≥ 5.0) in both virulent strains by comparison to all four attenuated strains. We examined biofilm formation and virulence in our OM model using a LAC mutant unable to produce one of these 14 proteins, specifically staphylocoagulase. The results confirmed that mutation of coa limits biofilm formation and, to a lesser extent, virulence in our OM model, although in both cases the limitation was reduced by comparison to the isogenic sarA mutant.