Expression Of fimA Research Articles

Identification of Critical Amino Acid Residues of a Two-Component Sensor Protein for Signal Sensing in Porphyromonas gingivalis Fimbriation via Random Mutant Library Construction.

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli-Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure-function relationships of proteins of interest.

Glyceryl Trinitrate, a Vasodilating Drug Acts as an Antibiofilm Agent in Serratia marcescens

Objectives: Serratia marcescens is a gram-negative pathogen of many species. The ability of S. marcescens to form biofilms and its potent innate resistance to antimicrobials and cleaning solutions are both essential for its pathogenicity and survival. The present study was conducted to investigate the effect of glyceryl trinitrate (GTN) on the biofilm of S. marcescens, as an alternative for antibiotic therapy.
 Methods: Different specimens, including ear swabs, burns, mid-stream urine, wound swabs, and sputum, were collected from patients who were brought to Al-Ramadi Hospital, Iraq. All samples were cultured, and the colonies that were obtained were detected using the VITEK® 2 compact. The ability of biofilms to develop was examined using the microtiter plate technique. The bactericidal effectiveness of GTN was estimated by the broth microdilution technique. The presence of fimA and fimC in S. marcescens isolates was detected using the polymerase chain reaction (PCR) method. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the effect of GTN on fimA and fimC gene expression. 
 Results: The results demonstrated that GTN has no effect on S. marcescens growth; while its biofilm was significantly (p<0.05) influenced. Moreover, all S. marcescens isolates had fimA and fimC, and the presence of GTN reduced the expression of these genes.
 Conclusion: The findings of this study reveal that GTN can act as a promising antibiofilm agent in reference to S. marcescens.

Cinnamaldehyde Resist Salmonella Typhimurium Adhesion by Inhibiting Type I Fimbriae.

Salmonella Typhimurium (S. Typhimurium), a common foodborne pathogen, severely harms the public and food security. Type I fimbriae (T1F) of S. Typhimurium, plays a crucial role in the pathogenic processes; it mediates the adhesion of bacteria to the mannose receptor on the host cell, assists the bacteria to invade the host cell, and triggers an inflammatory response. Cinnamaldehyde is the main ingredient in cinnamon essential oil. In this study, cinnamaldehyde was demonstrated to inhibit the expression of T1F by hemagglutination inhibition test, transmission electron microscopy, and biofilms. The mechanism of cinnamaldehyde action was studied by proteomics technology, PCR and Western blotting. The results showed that cinnamaldehyde can inhibit T1F in S. typhimurium without the growth of bacteria, by regulating the level of expression and transcription of fimA, fimZ, fimY, fimH and fimW. Proteomics results showed that cinnamaldehyde downregulated the subunits and regulators of T1F. In addition, the invasion assays proved that cinnamaldehyde can indeed reduce the ability of S. typhimurium to adhere to cells. The results of animal experiments showed that the colonization in the intestinal tract and the expression levels of inflammatory cytokine were significantly decreased, and the intestinal mucosal immune factors MUC1 and MUC2 were increased under cinnamaldehyde treatment. Therefore, cinnamaldehyde may be a potential drug to target T1F to treat Salmonella infections.

CRISPRi-mediated suppression of E. coli Nissle 1917 virulence factors: A strategy for creating an engineered probiotic using csgD gene suppression.

BackgroundBiofilm formation is a complex phenomenon, and it is the causative agent of several human infections. Bacterial amyloids are involved in biofilm formation leading to infection persistence. Due to antibiotic resistance, their treatment is a great challenge for physicians. Probiotics, especially E. coli Nissle 1917 (EcN), are used to treat human intestinal disorders and ulcerative colitis. It also expresses virulence factors associated with biofilm and amyloid formation. EcN produces biofilm equivalent to the pathogenic UPEC strains.MethodsCRISPRi was used to create the knockdown mutants of the csgD gene (csgD-KD). The qRT-PCR was performed to assess the expression of the csgD gene in csgD-KD cells. The csgD-KD cells were also evaluated for the expression of csgA, csgB, fimA, fimH, ompR, luxS, and bolA genes. The gene expression data obtained was further confirmed by spectroscopic, microscopic, and other assays to validate our study.ResultsCRISPRi-mediated knockdown of csgD gene shows reduction in curli amyloid formation, biofilm formation, and suppression of genes (csgA, csgB, fimA, fimH, ompR, bolA, and luxS) involved in virulence factors production.ConclusionCurli amyloid fibers and fimbriae fibers play a critical role in biofilm formation leading to pathogenicity. CsgD protein is the master regulator of curli synthesis in E. coli. Hence, curli amyloid inhibition through the csgD gene may be used to improve the EcN and different probiotic strains by suppressing virulence factors.

An in Vitro Study of Molecular Effects of a Combination Treatment with Antibiotics and Nanofluid Containing Carbon Nano-tubes on Klebsiella pneumoniae.

Background:We aimed to prepare a nanofluid, containing f-MWCNTs, and investigate the antibacterial efficacy of f-MWCNTs+ ciprofloxacin (cip) on Klebsiella pneumoniae by evaluating the virulence gene expression.Methods:This study was carried out from 2019 to 2020, in the Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran. The nanofluid containing antibiotic and f-MWCNTs were prepared by the ultrasonic method. The minimum inhibitory concentrations (MICs) of ciprofloxacin and f-MWCNTs were determined using the broth micro dilution MIC tests. For examining the antibacterial effects, the expression level of virulence genes, under the influence of f-MWCNTs, was evaluated by a real-time PCR.Results:The effect of 8 μg/ml ciprofloxacin + 400 μg/ml f-MWCNTs, completely inhibited the growth of the resistant isolate of K. pneumoniae, while, in the ATCC 700,603 isolate, 2 μg/ml ciprofloxacin with 100 μg/ml f-MWCNT could inhibit a bacterial growth. In the resistant K. pneumoniae clinical isolate, after f-MWCNT+cip treatment, the expression of fimA, fimD, wza, and wzi genes was significantly downregulated, compared to the ciprofloxacin treatment, and upregulated, compared to the negative control. For the ATCC 700,603 isolate treated with f-MWCNT+cip, the expression of fimA, fimD and wza virulence genes showed upregulation, compared to the negative control and downregulated in comparison with the ciprofloxacin treatment.Conclusion:Simultaneous treatment of resistant isolate of K. pneumoniae with f-MWCNTs +antibiotic could improve the effectiveness of antibiotic at lower doses, due to the reduced expression of virulence genes in comparison with antibiotic treatment, besides the increased cell wall permeability to antibiotics.

SRNA STnc150 is involved in virulence regulation of Salmonella Typhimurium by targeting fimA mRNA.

Small RNAs (sRNAs) are essential virulent regulators in Salmonella typhimurium (STM). To explore the role of sRNA STnc150 in regulating STM virulence, we constructed a STnc150 deletion strain (ΔSTnc150) and its complementary strain (ΔSTnc150/C). Then, we compared their characteristics to their original parent strain experimentally, identified the target genes of STnc150 and determined the expression levels of target genes. The results showed that the ΔSTnc150 strain exhibited delayed biofilm formation, enhanced adhesion to macrophages, significantly reduced LD50, increased liver and spleen viral loads and more vital pathological damaging ability than its parent and complementary strains. Further, bioinformatics combined with the bacterial dual plasmid reporter system confirmed that the bases 72-88 of STnc150 locating at the secondary stem-loop structure of the STnc150 are complementary with the bases 1-19 in the 5'-terminal of fimA mRNA of the type 1 fimbriae subunit. Western blot analysis showed that fimA protein level was increased in STnc150 strain compared with its parent and complementary strains. Together, this study suggested that STnc150 can down-regulate STM fimA expression at the translation level, which provided insights into the regulatory mechanisms of sRNAs in virulence of STM.

Exploring Photoactivated Disinfection-Induced Bystander Effects on Microbial Biofilms of Aggregatibacter actinomycetemcomitans.

Photoactivated disinfection (PAD) has been indicated as one of the potential methods for the initial periodontal therapy in periodontitis and peri-implantitis, which may have bystander effects on the viability of off-target cells. In this article, we evaluated the effects of PAD-induced bystander on biofilm formation ability, metabolic activity, and gene expression level of Aggregatibacter actinomycetemcomitans as one of the bacteria involved in causing periodontitis and peri-implantitis. Bystander effects induced by bacterial cell suspension (BCST) obtained from treated A. actinomycetemcomitans culture with indocyanine green (ICG)-PAD on biofilm formation of A. actinomycetemcomitans were determined using microbial viability assay. Additionally, metabolic activity and changes in gene expression of fimA were assessed using the XTT reduction method and quantitative real-time PCR (qRT-PCR) assay, respectively. The results revealed that 1000 and 125 μg/mL of ICG combined with diode laser irradiation for 1 min were significant and sub-significant reduction doses of ICG-PAD, respectively, versus A. actinomycetemcomitans. The biofilm formation ability, metabolic activity, as well as, expression level of fimA gene in A. actinomycetemcomitans were significantly reduced after treatment by a significant reduction dose of ICG-PAD-induced bystander effects (P<0.05). ICG-PAD could significantly reduce the microbial population, cell metabolic activity, and gene expression through the bystander effects and could enhance the efficiency of PAD as an adjunct approach for the nonsurgical treatment of periodontitis and peri-implantitis.

Xylitol Inhibits Growth and Blocks Virulence in Serratia marcescens

Serratia marcescens is an opportunistic nosocomial pathogen and causes wound and burn infections. It shows high resistance to antibiotics and its pathogenicity is mediated by an arsenal of virulence factors. Another therapeutic option to such infections is targeting quorum sensing (QS), which controls the expression of different S. marcescens virulence factors. Prevention of QS can deprive S. marcescens from its bacterial virulence without applying stress on the bacterial growth and facilitates the eradication of the bacteria by immunity. The objective of the current study is to explore the antimicrobial and antivirulence activities of xylitol against S. marcescens. Xylitol could inhibit the growth of S. marcescens. Sub-inhibitory concentrations of xylitol could inhibit biofilm formation, reduce prodigiosin production, and completely block protease activity. Moreover, xylitol decreased swimming motility, swarming motility and increased the sensitivity to hydrogen peroxide. The expression of rsmA, pigP, flhC, flhD fimA, fimC, shlA bsmB, and rssB genes that regulate virulence factor production was significantly downregulated by xylitol. In silico study showed that xylitol could bind with the SmaR receptor by hydrophobic interaction and hydrogen bonding, and interfere with the binding of the natural ligand with SmaR receptor. An in vivo mice survival test confirmed the ability of xylitol to protect mice against the virulence of S. marcescens. In conclusion, xylitol is a growth and virulence inhibitor in S. marcescens and can be employed for the treatment of S. marcescens wound and burn infections.

Efficacy of FimA antibody and clindamycin in silkworm larvae stimulated with Porphyromonas gulae

ABSTRACT Objective: Porphyromonas gulae, a major periodontal pathogen in animals, possesses fimbriae that have been classified into three genotypes (A, B, C) based on the diversity of fimA genes encoding fimbrillin protein (FimA). P. gulae strains with type C fimbriae were previously shown to be more virulent than other types. In this study, we further examined the host toxicity mediated by P. gulae fimbriae by constructing recombinant FimA (rFimA) expression vectors for each genotype and raised antibodies to the purified proteins. Methods and Results: All larvae died within 204 h following infection with P. gulae type C at the low-dose infection, whereas type A and B did not. Among fimA types, the survival rates of the larvae injected with rFimA type C were remarkably decreased, while the survival rates of the larvae injected with rFimA type A and type B were greater than 50%. Clindamycin treatment inhibited the growth of type C strains in a dose-dependent manner, resulting in an increased rate of silkworm survival. Finally, type C rFimA-specific antiserum prolonged the survival of silkworm larvae stimulated by infection with P. gulae type C strain or injection of rFimA type C protein. Conclusion: These results suggested that type C fimbriae have high potential for enhancement of bacterial pathogenesis, and that both clindamycin and anti-type C rFimA-specific antibodies are potent inhibitors of type C fimbriae-induced toxicity. This is the first report to establish a silkworm infection model using P. gulae for toxicity assessment.

Repurposing anti-diabetic drug "Sitagliptin" as a novel virulence attenuating agent in Serratia marcescens.

BackgroundSerratia marcescens is an emerging pathogen that causes a variety of health care associated infections. S. marcescens is equipped with an arsenal of virulence factors such as biofilm formation, swimming and swarming motilities, prodigiosin, protease and others which enable it to initiate and cause the infection. These virulence factors are orchestrated under the umbrella of an intercellular communication system named Quorum sensing (QS). QS allows bacterial population to synchronize the expression of virulence genes upon detection of a chemical signaling molecule. Targeting bacterial virulence is a promising approach to attenuate bacteria and enhances the ability of immune system to eradicate the bacterial infection. Drug repurposing is an advantageous strategy that confers new applications for drugs outside the scope of their original medical use. This promising strategy offers the use of safe approved compounds, which potentially lowers the costs and shortens the time than that needed for development of new drugs. Sitagliptin is dipeptidyl peptidase-4 (DPP-4) inhibitor, is used to treat diabetes mellitus type II as it increases the production of insulin and decreasing the production of glucagon by the pancreas. We aimed in this study to repurpose sitagliptin, investigating the anti-virulence activities of sitagliptin on S. marcescens.MethodsThe effect of sub-inhibitory concentrations of sitagliptin on virulence factors; protease, prodigiosin, biofilm formation, swimming and swarming motilities was estimated phenotypically. The qRT-PCR was used to show the effect of sitagliptin on the expression of QS-regulated virulence genes. The in-vivo protective activity of sitagliptin on S. marcescens pathogenesis was evaluated on mice.ResultsSitagliptin (1 mg/ml) significantly reduced the biofilm formation, swimming and swarming motilities, prodigiosin and protease. The qRT-PCR confirmed the effect on virulence as shown by down regulating the expression of fimA, fimC, flhC, flhD, bsmB, rssB, rsmA, pigP, and shlA genes. Moreover, the in-vivo findings showed the efficient ability of sitagliptin to weaken S. marcescens pathogenesis.ConclusionSitagliptin is a promising anti-virulence agent against S. marcescens that may be beneficial in the control of healthcare associated infections caused by S. marcescens.

Expression of virulence and antimicrobial resistance genes among Escherichia coli clinical isolates from blood and stool samples

Objective: As major nosocomial pathogens, Escherichia coli isolates exhibit antibiotic resistance and also express adhesive structures and antibiotic resistance genes. The objective of this study was the comparison of virulence gene expression of extended-spectrum beta-lactamase (ESBL)-producing E. coli between blood and stool samples. Methods: In this study, 20 E. coli clinical isolates (10 ESBL-producers including 5 from blood, 5 from stool samples and 10 non-ESBL-producer strains) were included. The existence of fimA, kpsMII and cdt (adhesives and toxin), acr-ab (efflux-encoding) and bla(CTX-M1) genes were confirmed by PCR. The quantitative real-time PCR was performed for evaluation of gene expression. Results: ESBL-producing E. coli isolates from stool samples could express fimA, kpsMII and cdt genes significantly higher than blood samples, whereas those isolates from blood samples significantly expressed the acr-ab (efflux-encoding) genes. In addition, the bla(CTXM1) gene was expressed among isolates from stool samples significantly higher (P = 0.022) than those from blood samples according to the analysis of variance (ANOVA) test. In addition, among non-ESBL-producers, the expression of fimA, kpsMII and cdt genes was significantly lower than ESBL-producing isolates from blood samples, but not significantly different than those from stool samples. Moreover, the expression of acr-ab genes was significantly lower than those from stool samples. Conclusion: The results exhibited that the expression of virulence genes among clinical isolates of E. coli is not the same or similar in various conditions or from various clinical origins. Thus determining the profile of gene expression in each of clinical situations can be helpful in tracking the infectious pathogens. ESBL-producing strains possibly have regulatory factors for inducing higher virulence gene expression. Copyright (C) 2019 Wolters Kluwer Health, Inc. All rights reserved.

Expression of adhesin genes and biofilm formation among Klebsiella oxytoca clinical isolates from patients with antibiotic-associated haemorrhagic colitis

Biofilm formation and resistance to last-line antibiotics have restricted chemotherapy options toward infection eradication. Fifty K. oxytoca isolates were collected from patients with antibiotic-associated haemorrhagic colitis (AAHC). Antibiotic susceptibility tests were conducted and phenotypic biofilm formation was assessed using microtitre tissue plate (MTP) assay. PCR was employed to amplify the adhesins, extended-spectrum β-lactamases (ESBLs), carbapenemase and colistin resistance genes. The expression of adhesin genes was evaluated using quantitative real-time PCR (RT-qPCR).Results/Key findings. The previous antibiotic consumption and hospitalization (P<0.05) and older ages (P=0.0033) were significantly associated with AAHC. None of the isolates produced biofilm strongly, but 70% of them produced moderate-level biofilm. The blaCTX-M (12/14), the blaIMP (8/14 MICIMI =4 µg ml-1 ) and blaOXA-48-like (5/14) and mcr-1 (4/14) genes were predominant, three of which harbouring all the genes. The expression of matB (0.023) and mrkA (0.011) was significantly different between multidrug-resistant and susceptible isolates. Furthermore, moderately biofilm producer isolates significantly exhibited higher expression of fimA (P=.0117), pilQ (P=0.002) and mrkA (P=0.020) genes compared to biofilm non-producers. No significant difference regarding gene expression was observed among ESBL alleles. Bacterial attachment by adhesins and biofilm formation among extensive drug-resistant K. oxytoca isolates hinder the efficient infection eradication. Hence, control and surveillance studies should be performed and other therapeutic auspicious approaches must be taken into account against AAHC, biofilm formation and drug resistance spread. Furthermore, previous antibiotic consumption and long-term hospitalization should be controlled.

Arginine within a specific motif near the N-terminal of FimY is critical for the maximal production of type 1 fimbriae in Salmonella enterica serovar Typhimurium.

An important Salmonella serovar for both human and animals Salmonella Typhimurium possesses 13 gene clusters that have the potential to produce fimbrial structure, among which the type 1 fimbriae with the binding specificity to mannose residue is the most commonly found type. Six structural genes and five regulatory genes comprise the fim gene cluster that is responsible for the production of type 1 fimbriae in S. Typhimurium. The fimY gene encodes a positive regulator for type 1 fimbrial expression since a deletion in fimY abolished the production of fimbriae. The N‐terminal portion of FimY contains amino acid residues that exhibit some similarity as those found in the proteins possessing the PilZ domain, which is engaged in cyclic di‐GMP binding. A fimY allele that had a change from arginine to alanine at position 7 (R7A) or 7 and 11 (R7/11A) generated by site‐directed mutagenesis in a 6RRERH11R motif near N‐terminal, when cloned in pACYC184 and transformed into a fimY‐deleted strain, decreased the expression of fimA and fimZ. The number of type 1 fimbriae in these two transformants was also less than those of the other transformants that contained different fimY alleles in pACYC184 when observed in electron microscopy. However, changing from arginine to alanine at position 11 (R11A) remained the same as the wild‐type fimY allele. It is likely that the arginine at the 7th position of FimY is critical for its maximal activating activity upon fimZ. Another motif 83DI85SLWIEK91G motif did not affect the function of FimY. Although FimY has the two aforementioned motifs, which contain some amino acids that are present within those of the PilZ domain proteins, secondary structure prediction analysis did not reveal that FimY has a conformation commonly observed in PilZ‐like proteins. Therefore, FimY and PilZ domain proteins are not homologs. Further investigation for a detailed analysis of FimY is thus warranted.

The regulation of Porphyromonas gingivalis biofilm formation by ClpP

Porphyromonas gingivalis is one of the most commonly detected pathogens in periodontal disease and root canal infections. Its viability and pathogenicity are greatly increased in plaque biofilms. Some caseinolytic proteases (Clp) reportedly regulate biofilm formation by various pathogenic bacteria, including P. gingivalis. However, the specific influence of ClpP and its mechanism of regulating biofilm formation by P. gingivalis remains unclear. Hence, in this study, a clpP deletion strain and complemented strain were constructed by homologous recombination, and an in vitro biofilm model was established. Biofilm architecture was observed by scanning electron microscopy. Bacterial cells within the biofilms were examined using confocal scanning laser microscopy. Crystal violet staining was used to determine the amount of formed biofilm. mRNA levels of related regulatory genes were assessed using real-time PCR. The clpP deletion and complemented strains of P. gingivalis were successfully constructed. The biofilm formation ability of the deletion strain was significantly reduced compared with that of the wild-type strain, while that of the complemented strain did not differ from that of the wild-type strain. The expression of fimA, mfa1, and luxS in the deletion strain was lower than in the wild-type and complemented strains at each timepoint. It can be concluded that ClpP increases the biofilm formation of P. gingivalis by regulating the expression levels of fimA, mfa1, and luxS.

Silymarin and curcumin effects on virulence and carbapenemase genes among multidrug-resistant Escherichia coli clinical isolates

Objective : Silymarin and curcumin antimicrobial properties have been previously described against some infectious agents. The aims of the current study were the investigation of the antibacterial effects of these compounds on the expression of fimA, cdt, bla IMP1 and bla OXA-48 and AcrAB-TolC genes among multidrug-resistant Escherichia coli clinical isolates. Methods : A total of five carbapenemase-producing E. coli strains were included. All of them carried bla IMP1 and bla OXA-48, ACR-AB, cdt, fimA and QepA genes. The antibiotic susceptibility test, imipenem, cefotaxime and ceftazidime minimum inhibitory concentration (MIC) and combine disk and Carba NP-test for carbapenemase production were performed. Silymarin and curcumin minimum inhibitory and bactericidal concentrations [MIC and minimum bactericidal concentration (MBC), respectively] were determined. The effect of 25 μg/ml concentration was also evaluated against gene expression. The quantitative real-time PCR was performed for the evaluation of genes expression. Results : Silymarin MIC and MBC were 256 and more than 256 μg/ml, respectively. Moreover, curcumin MIC and MBC concentrations were 128 and 256 μg/ml, respectively. Silymarin down-expressed the ACR-AB, cdt, fimA genes and QepA genes significantly (P &lt; 0.05), but conferred no significant effect on bla IMP nor bla OXA-48 genes. Moreover, curcumin down-expressed the ACR-AB, QepA and bla IMP genes significantly (P &lt; 0.05), but exhibited no significant effect against bla OXA-48, cdt and fimA genes. Conclusion : In this study, curcumin and silymarin sub-MIC concentrations could significantly inhibit the expression of the ACR-AB, cdt, fimA, QepA and bla IMP genes at 25 μg/ml. Combating bacterial virulence is a proper strategy not only to inhibit the spread of infections, but also to prevent the appearance and spread of antibiotic nonsusceptible strains.

Regulation of phase variation in type I pili formation in Escherichia coli: Role of alkylresorcinols, microbial autoregulators

Formation of virulence-associated type I pili in Escherichia coli should be considered as one of the most efficient models for investigating the mechanisms of regulating the heterogeneity of populations of genetically identical microbial cells. The present work focused on the role of alkylhydroxybenzenes (AHBs), density-dependent intercellular regulators, in controlling phase variations in type I pili formation (fimbriogenesis). The tested AHB homologue was C12-AHB; a genetically constructed strain E. сoli dsp250 containing the fimA-lacZ hybrid operon was used. In this operon, the fimA gene encodes the main subunit of the pili protein, and its expression results in β-galactosidase synthesis; pili-forming cells, therefore, become blue on the medium with the Х-gal substrate. Expression of fimA depends on the inversion of the fimS region that is located upstream of it. If the inversion is on, pili formation takes place, if it is off, no pili are formed. An increase in C12-AHB concentration (within the 5 × 10–5–2 × 10–4 M range) in the exponential-phase culture of strain dsp250 causes a dose-dependent change in the dominant phenotype that is displayed by up to 98–99% of the cells. Cells with this phenotype form colonies with a blue center and white edges. Up to 60% of the cells with this phenotype assume a metastable state and up to 11% and 44% of them transition to the alternative phenotypes of pili-forming and pili-less cells, respectively. The influence of C12-AHB on off-switching, i.e. the formation of the avirulent phenotype, was observed irrespective of the growth conditions of strain dsp250. Addition of glucose to the LB medium (5 or 10 mg/mL) resulted in catabolic repression via regulation by the cAMP-CNR complex and predictably induced pili formation in 49 and 75% of the cells, respectively. Against this background, C12-AHB caused a dose-dependent decrease in the share of pili-forming cells to 33–61% and an increase in the share of pili-less cells to 32–61%. If glucose was added in excess (2.5, 5 or 10 mg/mL) to the diluted LB/2 medium, pili formation was completely repressed, while C12-AHB still induced the off inversion to the pili-less phenotype in up to 30% of the cells. The conclusion can be drawn that C12-AHB is not involved in the pathway of fimbriogenesis regulation via cAMP. Since C12-AHB functions as an extracellular alarmon (activating the rpoS regulon and the SOS response as shown earlier, see Golod et al., 2009), its mechanism of action apparently involves stress signal transduction. It induces the synthesis of global regulators RpoS and H-NS and of intracellular alarmon (p) ppGpp; these factors are responsible for the on → off inversion and the proliferation of pili-less cells.

Gene expression profiling of fimA gene encoding fimbriae among clinical isolates of Porphyromonas gingivalis in response to photo-activated disinfection therapy

BackgroundThe endodontic disinfection therapy majorly aims to eradicate microbial pathogens. Photo-activated disinfection (PAD), also known as antimicrobial photodynamic therapy, is an alternative antimicrobial modality used to control the microorganisms causing endodontic infections. Notably, microorganisms may be exposed to sub-lethal doses of PAD (sPAD), influencing microbial virulence. The present study assessed the effects of sPAD on expression profiling of the gene associated with the biofilm formation being the most essential virulence factor in Porphyromonas gingivalis strain cells that survive the photodynamic reatment in vivo. Materialas and methodsSixteen clinical strains of PAD resistant P. gingivalis that were isolated in vivo, were further photosensitized in vitro with toluidine blue O (TBO), methylene blue (MB), and indocyanine green (ICG) as the photosensitizing agents, which were excited with specific wavelength of light based on the photosensitizer. After evaluating sPAD, its effects on the fimA gene expression were assessed using real-time quantitative reverse transcription PCR (qRT-PCR). ResultsIn this study, maximum values of sPAD against P. gingivalis were 6.25μg/mL TBO at a fluency of 171.87J/cm2, 15.6μg/mL ICG at fluency of 15.6J/cm2, and 25μg/mL MB at fluency of 93.75J/cm2. MB-, TBO-, and ICG-sPAD could cause about 4.6-, 14.4-, and 17.3-fold suppression of fimA expression, respectively. P. gingivalis strains expressed less virulence in cells surviving PAD. ConclusionsIn conclusion, the gene expression profiling was reduced in the bacterial cells, wherein greater reduction was observed for ICG-sPAD than TBO- and MB-sPAD.

FNR Regulates the Expression of Important Virulence Factors Contributing to the Pathogenicity of Avian Pathogenic Escherichia coli.

Avian pathogenic Escherichia coli (APEC) is the etiologic agent of colibacillosis, an important cause of morbidity and mortality in poultry. Though, many virulence factors associated with APEC pathogenicity are known, their regulation remains unclear. FNR (fumarate and nitrate reduction) is a well-known global regulator that works as an oxygen sensor and has previously been described as a virulence regulator in bacterial pathogens. The goal of this study was to examine the role of FNR in the regulation of APEC virulence factors, such as Type I fimbriae, and processes such as adherence and invasion, type VI secretion, survival during oxidative stress, and growth in iron-restricted environments. To accomplish this goal, APEC O1, a well-characterized, highly virulent, and fully sequenced strain of APEC harboring multiple virulence mechanisms, some of which are plasmid-linked, was compared to its FNR mutant for expression of various virulence traits. Deletion of FNR was found to affect APEC O1's adherence, invasion and expression of ompT, a plasmid-encoded outer membrane protein, type I fimbriae, and aatA, encoding an autotransporter. Indeed, the fnr− mutant showed an 8-fold reduction in expression of type I fimbriae and a highly significant (P < 0.0001) reduction in expression of fimA, ompT (plasmid-borne), and aatA. FNR was also found to regulate expression of the type VI secretion system, affecting the expression of vgrG. Further, FNR was found to be important to APEC O1's growth in iron-deficient media and survival during oxidative stress with the mutant showing a 4-fold decrease in tolerance to oxidative stress, as compared to the wild type. Thus, our results suggest that FNR functions as an important regulator of APEC virulence.

Role of yqiC in the Pathogenicity of Salmonella and Innate Immune Responses of Human Intestinal Epithelium.

The yqiC gene of Salmonella enterica serovar Typhimurium (S. Typhimurium) regulates bacterial growth at different temperatures and mice survival after infection. However, the role of yqiC in bacterial colonization and host immunity remains unknown. We infected human LS174T, Caco-2, HeLa, and THP-1 cells with S. Typhimurium wild-type SL1344, its yqiC mutant, and its complemented strain. Bacterial colonization and internalization in the four cell lines significantly reduced on yqiC depletion. Post-infection production of interleukin-8 and human β-defensin-3 in LS174T cells significantly reduced because of yqiC deleted in S. Typhimurium. The phenotype of yqiC mutant exhibited few and short flagella, fimbriae on the cell surface, enhanced biofilm formation, upregulated type-1 fimbriae expression, and reduced bacterial motility. Type-1 fimbriae, flagella, SPI-1, and SPI-2 gene expression was quantified using real-time PCR. The data show that deletion of yqiC upregulated fimA and fimZ expression and downregulated flhD, fliZ, invA, and sseB expression. Furthermore, thin-layer chromatography and high-performance liquid chromatography revealed the absence of menaquinone in the yqiC mutant, thus validating the importance of yqiC in the bacterial electron transport chain. Therefore, YqiC can negatively regulate FimZ for type-1 fimbriae expression and manipulate the functions of its downstream virulence factors including flagella, SPI-1, and SPI-2 effectors.

Salmonella grows vigorously on seafood and expresses its virulence and stress genes at different temperature exposure

BackgroundSeafood is not considered the natural habitat of Salmonella except the river fish, but still, the incidence of Salmonella in seafood is in a steady rise. By extending our understanding of Salmonella growth dynamics and pathogenomics in seafood, we may able to improve seafood safety and offer better strategies to protect the public health. The current study was thus aimed to assess the growth and multiplication of non-typhoidal and typhoidal Salmonella serovars on seafood and further sought to evaluate their virulence and stress genes expression while in contact with seafood at varying temperature exposure.ResultsSalmonella enterica Weltevreden and Salmonella enterica Typhi were left to grow on fish fillets at −20, 4, room temperature (RT) and 45 °C for a period of one week. Total RNA from both Salmonella serovars were extracted and qRT-PCR based relative gene expression approach was used to detect the expression of rpoE, invA, stn and fimA genes at four different temperature conditions studied on incubation days 0, 1, 3, 5 and 7. Salmonella Weltevreden growth on seafood was increased ~4 log10 at RT and 45 °C, nevertheless, nearly 2 and >4 log 10 reduction was observed in cell count stored at 4 and −20 °C on seafood, respectively. Growth pattern of Salmonella Typhi in seafood has shown identical pattern at RT and 45 °C, however, growth was sharply reduced at 4 and −20 °C as compared to the Salmonella Weltevreden. Total RNA of Salmonella Weltevreden was in the range from 1.3 to 17.6 μg/μl and maximum concentration was obtained at 45 °C on day 3. Similarly, RNA concentration of Salmonella Typhi was ranged from 1.2 to 11.8 μg/μl and maximum concentration was obtained at 45 °C on day 3. The study highlighted that expression of invA and stn genes of Salmonella Weltevreden was >8-fold upregulated at RT, whereas, fimA gene was increasingly down regulated at room temperature. Storage of Salmonella Weltevreden at 45 °C on seafood resulted in an increased expression (>13 -fold) of stn genes on day 1 followed by down regulation on days 3, 5, and 7. Nevertheless, other genes i.e. fimA, invA and rpo remained downregulated throughout the storage period. More intense upregulation was observed for invA and stn genes of Salmonella Typhi at RT and 45 °C. Further, incubating Salmonella Weltevreden at 4 °C resulted in down regulation in the expression of rpoE, invA and stn genes. Regarding Salmonella Typhi, fimA and stn genes were upregulated on day one, in addition, an increased expression of fimA was noted on day 3. At −20 °C, there was no obvious expression of target genes of Salmonella Weltevreden and Salmonella Typhi when stored along with seafood.ConclusionHere we demonstrate that nutritional constituents and water content available in seafood has become useful growth ingredients for the proliferation of Salmonella in a temperature dependent manner. Although, it was absence of serovar specific growth pattern of non-typhoidal and typhoidal Salmonella in seafood, there was observation of diverse expression profile of stress and virulent genes in non-typhoidal and typhoidal Salmonella serovars. In presence of seafood, the induced expression of Salmonella virulent genes at ambient temperature is most likely to be impacted by increased risk of seafood borne illness associated with Salmonella.