Bacterial Virulence Gene Expression Research Articles

Transcriptional responses of Neisseria gonorrhoeae to glucose and lactate: implications for resistance to oxidative damage and biofilm formation.

Understanding how bacteria adapt to different environmental conditions is crucial for advancing knowledge regarding pathogenic mechanisms that operate during infection as well as efforts to develop new therapeutic strategies to cure or prevent infections. Here, we investigated the transcriptional response of Neisseria gonorrhoeae, the causative agent of gonorrhea, to L-lactate and glucose, two important carbon sources found in the host environment. Our study revealed extensive transcriptional changes that gonococci make in response to L-lactate, with 37% of the gonococcal transcriptome being regulated, compared to only 9% by glucose. We found that L-lactate induces a transcriptional program that would negatively impact iron transport, potentially limiting the availability of labile iron, which would be important in the face of the multiple hydrogen peroxide attacks encountered by gonococci during its lifecycle. Furthermore, we found that L-lactate-mediated transcriptional response promoted aerobic respiration and dispersal of biofilms, contrasting with an anaerobic condition previously reported to favor biofilm formation. Our findings suggest an intricate interplay between carbon metabolism, iron homeostasis, biofilm formation, and stress response in N. gonorrhoeae, providing insights into its pathogenesis and identifying potential therapeutic targets.IMPORTANCEGonorrhea is a prevalent sexually transmitted infection caused by the human pathogen Neisseria gonorrhoeae, with ca. 82 million cases reported worldwide annually. The rise of antibiotic resistance in N. gonorrhoeae poses a significant public health threat, highlighting the urgent need for alternative treatment strategies. By elucidating how N. gonorrhoeae responds to host-derived carbon sources such as L-lactate and glucose, this study offers insights into the metabolic adaptations crucial for bacterial survival and virulence during infection. Understanding these adaptations provides a foundation for developing novel therapeutic approaches targeting bacterial metabolism, iron homeostasis, and virulence gene expression. Moreover, the findings reported herein regarding biofilm formation and L-lactate transport and metabolism contribute to our understanding of N. gonorrhoeae pathogenesis, offering potential avenues for preventing and treating gonorrhea infections.

Design, Synthesis, and Evaluation of New 1H-Benzo[d]imidazole Based PqsR Inhibitors as Adjuvant Therapy for Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor 1 (IC50 3.2 μM in P. aeruginosa PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled PpqsA-lux transcriptional reporter fusion in P. aeruginosa at low submicromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)-quinolones production.

Plankton and marine aggregates as transmission vectors for V. aestuarianus 02/041 infecting the pacific oyster Crassostrea gigas.

Vibrio aestuarianus is a bacterium related to mass mortality outbreaks of the Pacific oyster, Crassostrea gigas in Europe. In this study, the role of different planktonic substrates (phytoplankton cells, marine aggregates and chitin fragments) in mediating V. aestuarianus 02/041 infection of oysters was evaluated by controlled infection experiments. It was shown that phytoplankton cells and, to a greater extent, marine aggregates, significantly promote V. aestuarianus 02/041 intake by C. gigas maintained under stressful conditions in the laboratory. Such intake is associated with higher concentration of the pathogen in the bivalve hemolymph and compromised health status of infected oysters. In contrast, chitin particles do not play a significant role as transmission vector for V. aestuarianus 02/041 infecting its bivalve host. Interestingly, incorporation into marine aggregates foster extracellular proteases (ECPs) activity and a higher expression of bacterial virulence genes, that are potentially involved in bivalve infection. Results from this study contribute to elucidate transmission patterns of V. aestuarianus 02/041 to C. gigas that may be useful for the development of efficient measures to prevent and control oyster disease outbreaks.

Interplay between Immune and Bacterial Cells on a Biomimetic Nanostructured Surface: A "Race for the Surface" Study.

Biomaterial-associated infection is an ever-increasing risk with devasting consequences for patients. Considerable research has been undertaken to address this issue by imparting antibacterial properties to the surface of biomedical implants. One approach that generated much interest over recent years was the generation of bioinspired bactericidal nanostructures. In the present report, we have investigated the interplay between macrophages and bacteria on antibacterial nanostructured surfaces to determine the outcome of the so-called "race for the surface". Our results showed that macrophages can indeed outcompete Staphylococcus aureus via multiple mechanisms. The early generation of reactive oxygen species by macrophages, downregulation of bacterial virulence gene expression, and the bactericidal nature of the nanostructured surface itself collectively acted to help the macrophage to win the race. This study highlights the potential of nanostructured surfaces to reduce infection rates and improve the long-term success of biomedical implants. This work can also serve as guidance to others to investigate in vitro host-bacteria interactions on other candidate antibacterial surfaces.

PmiR senses 2-methylisocitrate levels to regulate bacterial virulence in Pseudomonas aeruginosa.

To adapt to changes in environmental cues, Pseudomonas aeruginosa produces an array of virulence factors to survive the host immune responses during infection. Metabolic products contribute to bacterial virulence; however, only a limited number of these signaling receptors have been explored in detail for their ability to modulate virulence in bacteria. Here, we characterize the metabolic pathway of 2-methylcitrate cycle in P. aeruginosa and unveil that PmiR served as a receptor of 2-methylisocitrate (MIC) to govern bacterial virulence. Crystallographic studies and structural-guided mutagenesis uncovered several residues crucial for PmiR's allosteric activation by MIC. We also demonstrated that PmiR directly repressed the pqs quorum-sensing system and subsequently inhibited pyocyanin production. Moreover, mutation of pmiR reduces bacterial survival in a mouse model of acute pneumonia infection. Collectively, this study identified P. aeruginosa PmiR as an important metabolic sensor for regulating expression of bacterial virulence genes to adapt to the harsh environments.

Protease Inhibitors from Solanum chacoense Inhibit Pectobacterium Virulence by Reducing Bacterial Protease Activity and Motility.

Potato is a major staple crop, and necrotrophic bacterial pathogens such as Pectobacterium spp. are a major threat to global food security. Most lines of cultivated potato (Solanum tuberosum) are susceptible to Pectobacterium spp., but some lines of wild potato are resistant, including Solanum chacoense M6. Despite the discovery of resistance in wild potatoes, specific resistance genes are yet to be discovered. Crude protein extract from M6 had a global effect on Pectobacterium brasiliense Pb1692 (Pb1692) virulence phenotypes. Specifically, M6 protein extracts resulted in reduced Pectobacterium exo-protease activity and motility, induced cell elongation, and affected bacterial virulence and metabolic gene expression. These effects were not observed from protein extracts of susceptible potato S. tuberosum DM1. A proteomics approach identified protease inhibitors (PIs) as candidates for S. chacoense resistance, and genomic analysis showed higher abundance and diversity of PIs in M6 than in DM1. We cloned five PIs that are unique or had high abundance in M6 compared with DM1 and purified the proteins (g18987, g28531, g39249, g40384, g6571). Four of the PIs significantly reduced bacterial protease activity, with strongest effects from g28531 and g6571. Three PIs (g18987, g28531, g6571) inhibited disease when co-inoculated with Pectobacterium pathogens into potato tubers. Two PIs (g28531, g6571) also significantly reduced Pb1692 motility and are promising as resistance genes. These results show that S. chacoense PIs contribute to bacterial disease resistance by inhibiting exo-proteases, motility, and tuber maceration and by modulating cell morphology and metabolism. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Fabrication of Isopropanolamine-Decorated Coumarin Derivatives as Novel Quorum Sensing Inhibitors to Suppress Plant Bacterial Disease.

Emerging pesticide-resistant phytopathogenic bacteria have become a stumbling block in the development and use of pesticides. Quorum sensing (QS) blockers, which interfere with bacterial virulence gene expression, are a compelling way to manage plant bacterial disease without resistance. Herein, a series of isopropanolamine-decorated coumarin derivatives were designed and synthesized, and their potency in interfering with QS was investigated. Notably, compound A5 exhibited a better bioactivity with median effective concentration (EC50) values of 6.75 mg L-1 against Xanthomonas oryzae pv. oryzae (Xoo) than bismerthiazol (EC50 = 21.9 mg L-1). Further biochemical studies revealed that compound A5 disturbed biofilm formation and suppressed bacterial virulence factors and so forth. Moreover, compound A5 decreased the expression of QS-related genes. Interestingly, compound A5 had the acceptable control effect (53.2%) toward Xoo in vivo. Overall, this study identifies a novel lead compound for the development of bactericide candidates to control plant bacterial diseases by interfering with QS.

Temporal Transcriptional Responses of a Vibrio alginolyticus Strain to Podoviridae Phage HH109 Revealed by RNA-Seq.

ABSTRACTPhage are thought to exhibit control over host genes during infection. As a preliminary investigation of the kinetics and magnitude of co-expression between phage and bacteria, we compared the global transcriptional profiles for Vibrio alginolyticus strain E110 and its lytic phage HH109 by using RNA sequencing. In total, 24.7% (1,143/4,620) of the host protein-coding genes were differentially expressed genes during infection (DEGs). Functional analysis of the host DEGs suggests that phage HH109 induced rapid and distinctive changes when compared with 60- and 120-min postinfection (mpi). Based on gene co-expression network analysis, an uncharacterized late gene gp27 encoded by the phage HH109 was predicted to modulate the host’s membrane transport and/or transcriptional regulation. Furthermore, expression of several bacterial virulence genes was downregulated while drug resistance genes were upregulated. This work contributes to an in-depth understanding of the reciprocal interactions of lytic phage HH109 and its pathogenic Vibrio host E110, and can provide new insights into the research and development of phage therapy against pathogenic Vibrio infections in the economically significant aquatic animals.IMPORTANCE Vibrio alginolyticus is a common opportunistic pathogen that causes mass mortality in cultured marine animals. Phage HH109 lyses pathogenic V. alginolyticus strain E110 with high efficiency and thus serves as a useful model to understand the dynamic interplay of a phage and its host. Global transcriptomic responses of strain E110 post-HH109 infection were characterized by using RNA sequencing, elucidating step-by-step control by HH109, an antiphage-like responses, and the elevated expression of drug resistance. This study provides a detailed molecular description phage and V. alginolyticus, providing insight into better prevention and control of vibriosis in aquatic animals.

Abstract 9952: Periodontal Inflammation, Periodontal Bacterial Load and Virulence Genes Expression Are Risk Factors for Myocardial Infarction

Background: A causal relationship between periodontitis and Myocardial Infarction (MI) is stipulated but remains incompletely characterised. Methods: Prospectively recruited 160 acute MI patients underwent periodontal exam to quantify periodontal inflamed surface area (PISA), probing pocket depths (PPD) and clinical attachment loss (CAL). Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans and Prevotella intermedia loads were assessed in subgingival plaque, venous blood, aspirated intra-coronary thrombi and deployment balloons using quantitative polymerase chain reaction. Expression of periodontal bacterial virulence genes (n=13) was quantified with reverse transcription PCR. Myocardial injury was estimated by 12-hour troponin (TnI) and severity of coronary artery disease (CAD) burden by SYNTAX-I scores. CAD-symptom free controls (n=50) of similar age/gender distribution were used for comparison. Results: Compared to controls, MI patients had worse periodontitis and higher bacterial loads (all p <0.05). In MI cohort, after adjusting established risk factors, hierarchical linear regression showed that PISA (5%), mean PPD (3%), mean CAL (2%), P. gingivalis load (4%) and upregulated expression of bioF-3 , fimA , prtH , prtP , ltxA , cdtB virulence genes (4-9%) were associated with an increase in Tnl release, all p <0.05. Similarly, mean PPD (5%), mean CAL (4%), P. gingivalis load (5%) and upregulated expression of bioF-3 , fimA , prtP and ltxA virulence genes (4-9%) were associated with higher SYNTAX-I score, all p <0.05. Every 200 mm 2 PISA, 1mm mean PPD, 1mm mean CAL and 10 9 P. gingivalis copies/ng DNA was responsible for 19,000, 32,000, 38,000 and 10,000 ng/L increase in TnI release, respectively. The SYNTAX-I score increased by 3, 4 and 1 unit with every 1mm mean PPD, 1mm mean CAL and 10 9 P. gingivalis copies/ng DNA, respectively. Periodontal bacterial DNA was detected in coronary thrombi and subgingival plaque sampled from the same patients, with a strong correlation for P. gingivalis and T. forsythia bacterial loads (Spearman's rho=0.6 for both, p <0.05). Conclusions: These findings enable further recognition of periodontitis as a risk factor for MI; a new candidate for primary and secondary prevention.

In silico Evaluation of Savirin Derivatives As Inhibitors of the agr Quorum Sensing System of Staphylococcus aureus

Bacterial resistance to current antibacterial agents is a global challenge. The problem of bacterial resistance can be addressed by targeting the bacterial virulence gene expression, particularly quorum sensing (QS) mechanism which is cell to cell communication. Expression of various virulence genes is regulated by the agr QS system of S. aureus, which can be attenuated by inhibiting AgrA-DNA interactions. In this paper molecular docking study was done for savirin and its thirty derivatives with AgrA protein (PDB ID: 4G4K) of S. aureus in order to find out potential inhibitors of AgrA DNA binding activity. Out of the thirty derivatives of savirin, compounds 4, 7, 17, 20, 26 and 30 showed excellent binding scores (less than -10 kcal/mol). Molecular dynamics simulations of the above protein-ligand complexes along with savirin suggested that the complexes of compounds 4, 7, 17, 20, 26 and 30 are stable. MM/PB(GB)SA binding energy calculation showed that compounds 20 and 30 have higher binding affinity among the other compounds. Density functional theory based electronic properties of the compounds were also calculated. Compounds 20 and 30 possess very good drug likeness properties and hence, can be used as potential inhibitors against agr QS system of S. aureus.

Antibiofilm and antivirulence potential of silver nanoparticles against multidrug-resistant Acinetobacter baumannii

We aimed to isolate Acinetobacter baumannii (A. baumannii) from wound infections, determine their resistance and virulence profile, and assess the impact of Silver nanoparticles (AgNPs) on the bacterial growth, virulence and biofilm-related gene expression. AgNPs were synthesized and characterized using TEM, XRD and FTIR spectroscopy. A. baumannii (n = 200) were isolated and identified. Resistance pattern was determined and virulence genes (afa/draBC, cnf1, cnf2, csgA, cvaC, fimH, fyuA, ibeA, iutA, kpsMT II, PAI, papC, PapG II, III, sfa/focDE and traT) were screened using PCR. Biofilm formation was evaluated using Microtiter plate method. Then, the antimicrobial activity of AgNPs was evaluated by the well-diffusion method, growth kinetics and MIC determination. Inhibition of biofilm formation and the ability to disperse biofilms in exposure to AgNPs were evaluated. The effect of AgNPs on the expression of virulence and biofilm-related genes (bap, OmpA, abaI, csuA/B, A1S_2091, A1S_1510, A1S_0690, A1S_0114) were estimated using QRT-PCR. In vitro infection model for analyzing the antibacterial activity of AgNPs was done using a co-culture infection model of A. baumannii with human fibroblast skin cell line HFF-1 or Vero cell lines. A. baumannii had high level of resistance to antibiotics. Most of the isolates harbored the fimH, afa/draBC, cnf1, csgA and cnf2, and the majority of A. baumannii produced strong biofilms. AgNPs inhibited the growth of A. baumannii efficiently with MIC ranging from 4 to 25 µg/ml. A. baumannii showed a reduced growth rate in the presence of AgNPs. The inhibitory activity and the anti-biofilm activity of AgNPs were more pronounced against the weak biofilm producers. Moreover, AgNPs decreased the expression of kpsMII , afa/draBC,bap, OmpA, and csuA/B genes. The in vitro infection model revealed a significant antibacterial activity of AgNPs against extracellular and intracellular A. baumannii. AgNPs highly interrupted bacterial multiplication and biofilm formation. AgNPs downregulated the transcription level of important virulence and biofilm-related genes. Our findings provide an additional step towards understanding the mechanisms by which sliver nanoparticles interfere with the microbial spread and persistence.

Host–Microbe Interactions: Wallowing in Mucus Mire

Mucin glycoproteins confer robust protection against infection by regulating bacterial behavior and virulence gene expression, but the mechanistic bases are poorly understood. New work implicates glycan-based signaling through a carbohydrate-binding sensor kinase in mediating pathogen behavior at the mucosal interface.

Interaction of human oral cancer and the expression of virulence genes of dental pathogenic bacteria

Oral squamous cell carcinoma (OSCC) are one of the major causes of cancer morbidity and mortality worldwide. Dental microbiome has been considered as inducing agents in oral carcinogenesis. Therefore, the objective of this study was to investigate the interaction of the gene expression of the dental microbiome and OSCC patients. A cross-sectional study was designed by recruiting confirmed OSCC patients attending the University hospital during October 2018 and July 2019. The dental bacteria were isolated and confirmed by PCR technique. The expression of host and bacterial virulence genes was determined using qPCR. This study shows that 54% of T. forsythia found to be the most predominant organisms in 30 positive cases, followed by 34% of Campylobacter rectus and 29% of Prevotella intermedia. The expression of mRNA levels of bspA, csxA, fadA and interpain A in the OSCC- bacteria positive cases was significantly higher than the control group (P < 0.001). It was further found that interpainA, csxA, fadA, and bspA genes have the potential effects on the cellular gene expression in OSCC patients. A significant correlation was seen between expression patterns of CXCL10, DIAPH1, NCLN and MMP9 genes with interpain A, fadA, and bspA involved in OSCC cases The results indicate that the species specific bacteria may play a role in triggering chronic inflammation in OSCC patients. Therefore, alteration in the gene expression through the dental microbiome could be used as an alternative target in the clinical practice to detect OSCC.

Enzyme-assisted extraction of flavanones from citrus pomace: Obtention of natural compounds with anti-virulence and anti-adhesive effect against Salmonella enterica subsp. enterica serovar Typhimurium

Food poisoning has been recognized as a global challenge, causing illness in millions of people. Salmonella enterica subsp. enterica serovar Typhimurium is a major foodborne pathogen causing disease in humans by expressing virulence genes to invade the host epithelial cells. Thus, the search for natural compounds with anti-virulence and anti-adhesive effect could help to mitigate Salmonella infection. This study aimed to determine the effect of flavanones on S. Typhimurium virulence and adhesion to Caco-2 cells. Flavanones were obtained from citrus pomace (CP) by enzyme-assisted (CP-E) and conventional hydroalcoholic (CP–H) extraction methods, which produced extracts rich in aglycones and glycosylated flavanones, respectively. CP-E and aglycone standards (naringenin and hesperetin) decreased bacterial growth (25–90%), motility (50–90%), and adhesion to Caco-2 cells (25–50%). CP-H and glycosylated flavanones (naringin and hesperidin) affected only bacterial adhesion (decreased 20–75%). Agglutination assays demonstrated the binding of extracts and pure flavanones to S. Typhimurium adhesion structures. Fimbrial gene expression (fimA and fimZ) was downregulated in the presence of CP-H, CP-E, and aglycones. Both CP extracts decreased the expression of genes associated with cell invasion (hilA, ssrB and sopD). These results suggest that CP extracts and flavanones inhibit S. Typhimurium adhesion by interacting with fimbriae and flagella structures and downregulating fimbrial and virulence genes. CP-E and aglycone flavanones, rarely found in nature, exhibited superior inhibitory effects on bacterial viability, motility, adhesion and virulence gene expression than glycosylated flavanones. The outcomes of this research advance the understanding of how aglycone and glycosylated flavanones impact S. Typhimurium virulence activity and adhesion, contributing to their application as functional food ingredients for the prevention of adhesion, invasion, and the development of this pathogen in the gut.

Dual Role of Auxin in Regulating Plant Defense and Bacterial Virulence Gene Expression During Pseudomonas syringae PtoDC3000 Pathogenesis.

Modification of host hormone biology is a common strategy used by plant pathogens to promote disease. For example, the bacterial pathogen strain Pseudomonas syringae DC3000 (PtoDC3000) produces the plant hormone auxin (indole-3-acetic acid [IAA]) to promote PtoDC3000 growth in plant tissue. Previous studies suggest that auxin may promote PtoDC3000 pathogenesis through multiple mechanisms, including both suppression of salicylic acid (SA)-mediated host defenses and via an unknown mechanism that appears to be independent of SA. To test if host auxin signaling is important during pathogenesis, we took advantage of Arabidopsis thaliana lines impaired in either auxin signaling or perception. We found that disruption of auxin signaling in plants expressing an inducible dominant axr2-1 mutation resulted in decreased bacterial growth and that this phenotype was suppressed by introducing the sid2-2 mutation, which impairs SA synthesis. Thus, host auxin signaling is required for normal susceptibility to PtoDC3000 and is involved in suppressing SA-mediated defenses. Unexpectedly, tir1 afb1 afb4 afb5 quadruple-mutant plants lacking four of the six known auxin coreceptors that exhibit decreased auxin perception, supported increased levels of bacterial growth. This mutant exhibited elevated IAA levels and reduced SA-mediated defenses, providing additional evidence that auxin promotes disease by suppressing host defense. We also investigated the hypothesis that IAA promotes PtoDC3000 virulence through a direct effect on the pathogen and found that IAA modulates expression of virulence genes, both in culture and in planta. Thus, in addition to suppressing host defenses, IAA acts as a microbial signaling molecule that regulates bacterial virulence gene expression.

Molecular Mechanisms of Phosphate Stress Activation of Pseudomonas aeruginosa Quorum Sensing Systems.

The hierarchical quorum sensing (QS) systems of Pseudomonas aeruginosa, consisting of las, pqs, and rhl, coordinate the expression of bacterial virulence genes. Previous studies showed that under phosphate deficiency conditions, two-component regulatory system PhoRB could activate various genes involved in cytotoxicity through modulation of QS systems, but the mechanism by which PhoR/PhoB influences QS remains largely unknown. Here, we provide evidence that among the key QS regulatory genes in P. aeruginosa, rhlR, pqsA, mvfR, and lasI were activated by the response regulator PhoB under phosphate-depleted conditions. We show that PhoB is a strong competitor against LasR and RsaL for binding to the promoter of lasI and induces significant expression of lasI, rhlR, and mvfR. However, expression of lasI, encoding the signal 3-oxo-C12-HSL, was increased only marginally under the same phosphate-depleted conditions. This seeming inconsistency was attributed to the induction of pvdQ, which encodes an enzyme for degradation of 3-oxo-C12-HSL signal molecules. Taken together, the results from this study demonstrate that through the two-component regulatory system PhoR/PhoB, phosphate depletion stress could influence the QS network by modulating several key regulators, including lasI, rhlR, mvfR, and pvdQ The findings highlight not only the potency of the PhoR/PhoB-mediated bacterial stress response mechanism but also the plasticity of the P. aeruginosa QS systems in coping with the changed environmental conditions.IMPORTANCE It is not fully understood how phosphate deficiency could influence the virulence of Pseudomonas aeruginosa through modulation of the bacterial QS systems. This report presents a systemic investigation on the impact of phosphate depletion on the hierarchy of quorum sensing systems of P. aeruginosa The results showed that phosphate stress could have an extensive impact on the QS networks of this bacterial pathogen. Among the 7 QS regulatory genes representing the 3 sets of QS systems tested, 4 were significantly upregulated by phosphate depletion stress through the PhoR/PhoB two-component regulatory system, especially the upstream QS regulatory gene lasI We also present evidence that the response regulator PhoB was a strong competitor against the las regulators LasR and RsaL for the lasI promoter, unveiling the mechanistic basis of the process by which phosphate stress could modulate the bacterial QS systems.

Anti-staphylococcus Antibiotics Interfere With the Transcription of Leucocidin ED Gene in Staphylococcus aureus Strain Newman.

Antibiotics have been described to modulate bacterial virulence gene expression. This study aimed to assess the changes caused by anti-Staphylococcus agents in the transcription of leucocidin ED (lukED) gene of Staphylococcus aureus strain Newman in vitro and in vivo and to determine whether the altered expression is agr dependent. The bacteria were exposed to subinhibitory concentrations [1/2, 1/4, or 1/8 minimal inhibitory concentration (MIC)] of 11 antibiotics, and the expression of lukE and agr-effector RNAIII was determined using qRT-PCR. In vivo experiments were performed to evaluate the impact exerted by six representative antibiotics on the transcription of both genes. Molecular analysis showed that in vitro lukE transcription was dramatically promoted in the Newman strain exposed to sub-MICs of vancomycin, trimethoprim–sulfamethoxazole, clindamycin, gentamicin, daptomycin, and ciprofloxacin and considerably reduced when stimulated by cefazolin, erythromycin, rifampicin, tigecycline, and linezolid. In the murine abscess model, tigecycline significantly decreased the transcription of lukE and the bacterial numbers, whereas vancomycin increased them; although cefazolin increased the lukE expression (contrary to the in vitro effect), it had a remarkable role in reducing bacterial load. The correspondence analysis shows that RNAIII expression varied under seven of 11 antibiotics in vitro, and six drugs in vivo were consistent with lukE transcripts. In conclusion, our data show that anti-Staphylococcus antibiotics exert modulatory effects on lukE expression in vitro and/or in vivo, and the changed expression caused by some drugs may be involved with agr activity, thus providing a guide to choose appropriate agents to avoid promoting bacterial virulence in lukED-positive S. aureus infections.

Indole Signaling at the Host-Microbiota-Pathogen Interface.

Microbial establishment within the gastrointestinal (GI) tract requires surveillance of the gut biogeography. The gut microbiota coordinates behaviors by sensing host- or microbiota-derived signals. Here we show for the first time that microbiota-derived indole is highly prevalent in the lumen compared to the intestinal tissue. This difference in indole concentration plays a key role in modulating virulence gene expression of the enteric pathogens enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium Indole decreases expression of genes within the locus of enterocyte effacement (LEE) pathogenicity island, which is essential for these pathogens to form attaching and effacing (AE) lesions on enterocytes. We synthetically altered the concentration of indole in the GI tracts of mice by employing mice treated with antibiotics to deplete the microbiota and reconstituted with indole-producing commensal Bacteroides thetaiotaomicron (B. theta) or a B. theta ΔtnaA mutant (does not produce indole) or by engineering an indole-producing C. rodentium strain. This allowed us to assess the role of self-produced versus microbiota-produced indole, and the results show that decreased indole concentrations promote bacterial pathogenesis, while increased levels of indole decrease bacterial virulence gene expression. Moreover, we identified the bacterial membrane-bound histidine sensor kinase (HK) CpxA as an indole sensor. Enteric pathogens sense a gradient of indole concentrations in the gut to probe different niches and successfully establish an infection.IMPORTANCE Pathogens sense and respond to several small molecules within the GI tract to modulate expression of their virulence repertoire. Indole is a signaling molecule produced by the gut microbiota. Here we show that indole concentrations are higher in the lumen, where the microbiota is present, than in the intestinal tissue. The enteric pathogens EHEC and C. rodentium sense indole to downregulate expression of their virulence genes, as a read-out of the luminal compartment. We also identified the bacterial membrane-bound HK CpxA as an indole sensor. This regulation ensures that EHEC and C. rodentium express their virulence genes only at the epithelial lining, which is the niche they colonize.

Dual RNA-Seq Unveils Pseudomonas plecoglossicida htpG Gene Functions During Host-Pathogen Interactions With Epinephelus coioides.

Pseudomonas plecoglossicida is a temperature-dependent opportunistic pathogen which is associated with a variety of diseases in fish. During the development of “white nodules” disease, the expression of htpG in P. plecoglossicida was found to be significantly up-regulated at its virulent temperature of 18°C. The infection of htpG-RNAi strain resulted in the onset time delay, reduction in mortality and infection symptoms in spleen of Epinephelus coioides, and affected the bacterial tissue colonization. In order to reveal the effect of htpG silencing of P. plecoglossicida on the virulence regulation in P. plecoglossicida and immune response in E. coioides, dual RNA-seq was performed and a pathogen-host integration network was constructed. Our results showed that infection induced the expression of host genes related to immune response, but attenuated the expression of bacterial virulence genes. Novel integration was found between host immune genes and bacterial virulence genes, while IL6, IL1R2, IL1B, and TLR5 played key roles in the network. Further analysis with GeneMANIA indicated that flgD and rplF might play key roles during the htpG-dependent virulence regulation, which was in accordance with the reduced biofilm production, motility and virulence in htpG-RNAi strain. Meanwhile, IL6 and IL1B were found to play key roles during the defense against P. plecoglossicida, while CELA2, TRY, CPA1, CPA2, and CPB1 were important targets for P. plecoglossicida attacking to the host.

A versatile remote control system for functional expression of bacterial virulence genes based on the tetA promoter

The expression of bacterial virulence factors is controlled in response to host or environmental factors and most virulence genes are not expressed under laboratory conditions. Investigations of molecular structures and cellular functions of bacterial virulence factors demand systems for experimentally controlled expression. We describe a simple and robust system that is based on the tetA promoter and the cognate repressor TetR. Expression under control of PtetA can be induced by non-antibiotic derivatives of tetracycline such as anhydrotetracycline (AHT). Tet-on expression cassettes can be used to replace native promoters of chromosomal genes or operons of interest. Tet-on plasmids allow episomal expression in homologous or heterologous host organisms. We demonstrate the application of Tet-on systems for the controlled induction of flagella assembly and motility, and for surface expression of adhesins of the chaperone/usher family of enteropathogenic Escherichia coli and autotransporter adhesins of Yersinia enterocolitica in Salmonella enterica and E. coli. Since inducer AHT can easily cross bacterial envelopes and mammalian cell membranes, the system can also be applied to control virulence genes in intracellular bacteria. We demonstrate the controlled synthesis, translocation and function of effector proteins of the type III secretion system of intracellular S. enterica.