scholarly journals Inactivation of Genes Encoding MutL and MutS Proteins Influences Adhesion and Biofilm Formation by Neisseria gonorrhoeae

2019 ◽  
Vol 7 (12) ◽  
pp. 647 ◽  
Author(s):  
Jagoda Płaczkiewicz ◽  
Monika Adamczyk-Popławska ◽  
Robert Lasek ◽  
Pawel Bącal ◽  
Agnieszka Kwiatek
Keyword(s):  
Epithelial Cells ◽  
Neisseria Gonorrhoeae ◽  
Biofilm Formation ◽  
Dna Microarrays ◽  
Wild Type ◽  
Human Genes ◽  
Genes Encoding ◽  
Differential Gene ◽  
Bacterial Genes ◽  
Muts Gene

Neisseria gonorrhoeae is an etiological agent of gonorrhea, which remains a global health problem. This bacterium possesses MutL and MutS DNA repair proteins encoded by mutL and mutS genes, whose inactivation causes a mutator phenotype. We have demonstrated the differential gene expression in N. gonorrhoeae mutL and mutS mutants using DNA microarrays. A subset of differentially expressed genes encodes proteins that can influence adhesion and biofilm formation. Compared to the wild-type strain, N. gonorrhoeae mutL and mutS mutants formed denser biofilms with increased biofilm-associated biomass on the abiotic surface. The N. gonorrhoeae mutS::km, but not the mutL mutant, was also more adherent and invasive to human epithelial cells. Further, during infection of epithelial cells with N. gonorrhoeae mutS::km, the expression of some bacterial genes encoding proteins that can influence gonococcal adhesion was changed compared with their expression in cells infected with the wild-type gonococcus, as well as of human genes’ encoding receptors utilized by N. gonorrhoeae (CD46, CEACAM 1, HSPG 2). Thus, deficiency in the mutS gene resulting in increased mutation frequency in singular organisms can be beneficial in populations because these mutants can be a source of features linked to microbial fitness.

scholarly journals Evaluation of the effects of sdiA, a luxR homologue, on adherence and motility of Escherichia coli O157 : H7

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1303-1312 ◽  
Author(s):  
Vijay K. Sharma ◽  
Shawn M. D. Bearson ◽  
Bradley L. Bearson
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Mutant Strain ◽  
Regulatory Networks ◽  
Double Mutant ◽  
Negative Regulator ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Expression Of Genes ◽  
Genes Encoding

Quorum-sensing (QS) signalling pathways are important regulatory networks for controlling the expression of genes promoting adherence of enterohaemorrhagic Escherichia coli (EHEC) O157 : H7 to epithelial cells. A recent study has shown that EHEC O157 : H7 encodes a luxR homologue, called sdiA, which upon overexpression reduces the expression of genes encoding flagellar and locus of enterocyte effacement (LEE) proteins, thus negatively impacting on the motility and intimate adherence phenotypes, respectively. Here, we show that the deletion of sdiA from EHEC O157 : H7 strain 86-24, and from a hha (a negative regulator of ler) mutant of this strain, enhanced bacterial adherence to HEp-2 epithelial cells of the sdiA mutant strains relative to the strains containing a wild-type copy of sdiA. Quantitative reverse transcription PCR showed that the expression of LEE-encoded genes ler, espA and eae in strains with the sdiA deletions was not significantly different from that of the strains wild-type for sdiA. Similarly, no additional increases in the expression of LEE genes were observed in a sdiA hha double mutant strain relative to that observed in the hha deletion mutant. While the expression of fliC, which encodes flagellin, was enhanced in the sdiA mutant strain, the expression of fliC was reduced by several fold in the hha mutant strain, irrespective of the presence or absence of sdiA, indicating that the genes sdiA and hha exert opposing effects on the expression of fliC. The strains with deletions in sdiA or hha showed enhanced expression of csgA, encoding curlin of the curli fimbriae, with the expression of csgA highest in the sdiA hha double mutant, suggesting an additive effect of these two gene deletions on the expression of csgA. No significant differences were observed in the expression of the genes lpfA and fimA of the operons encoding long polar and type 1 fimbriae in the sdiA mutant strain. These data indicate that SdiA has no significant effect on the expression of LEE genes, but that it appears to act as a strong repressor of genes encoding flagella and curli fimbriae, and the alleviation of the SdiA-mediated repression of these genes in an EHEC O157 : H7 sdiA mutant strain contributes to enhanced bacterial motility and increased adherence to HEp-2 epithelial cells.

scholarly journals CD46 is phosphorylated at tyrosine 354 upon infection of epithelial cells by Neisseria gonorrhoeae

2002 ◽  
Vol 156 (6) ◽  
pp. 951-957 ◽  
Author(s):  
Shaun W. Lee ◽  
Robert A. Bonnah ◽  
Dustin L. Higashi ◽  
John P. Atkinson ◽  
Sharon L. Milgram ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Neisseria Gonorrhoeae ◽  
Tyrosine Phosphorylation ◽  
Bacterial Adhesion ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Host Cells ◽  
Cell Cortex ◽  
Wild Type

The Neisseria type IV pilus promotes bacterial adhesion to host cells. The pilus binds CD46, a complement-regulatory glycoprotein present on nucleated human cells (Källström et al., 1997). CD46 mutants with truncated cytoplasmic tails fail to support bacterial adhesion (Källström et al., 2001), suggesting that this region of the molecule also plays an important role in infection. Here, we report that infection of human epithelial cells by piliated Neisseria gonorrhoeae (GC) leads to rapid tyrosine phosphorylation of CD46. Studies with wild-type and mutant tail fusion constructs demonstrate that Src kinase phosphorylates tyrosine 354 in the Cyt2 isoform of the CD46 cytoplasmic tail. Consistent with these findings, infection studies show that PP2, a specific Src family kinase inhibitor, but not PP3, an inactive variant of this drug, reduces the ability of epithelial cells to support bacterial adhesion. Several lines of evidence point to the role of c-Yes, a member of the Src family of nonreceptor tyrosine kinases, in CD46 phosphorylation. GC infection causes c-Yes to aggregate in the host cell cortex beneath adherent bacteria, increases binding of c-Yes to CD46, and stimulates c-Yes kinase activity. Finally, c-Yes immunoprecipitated from epithelial cells is able to phosphorylate the wild-type Cyt2 tail but not the mutant derivative in which tyrosine 354 has been substituted with alanine. We conclude that GC infection leads to rapid tyrosine phosphorylation of the CD46 Cyt2 tail and that the Src kinase c-Yes is involved in this reaction. Together, the findings reported here and elsewhere strongly suggest that pilus binding to CD46 is not a simple static process. Rather, they support a model in which pilus interaction with CD46 promotes signaling cascades important for Neisseria infectivity.

scholarly journals A Genome-Wide Screen Identifies Genes in Rhizosphere-AssociatedPseudomonasRequired to Evade Plant Defenses

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Zhexian Liu ◽  
Polina Beskrovnaya ◽  
Ryan A. Melnyk ◽  
Sarzana S. Hossain ◽  
Sophie Khorasani ◽  
...  
Keyword(s):  
Plant Growth ◽  
Biofilm Formation ◽  
Plant Defenses ◽  
Plant Root ◽  
Plant Health ◽  
Wild Type ◽  
Content Type ◽  
Host Immune Responses ◽  
A Genome ◽  
Bacterial Genes

ABSTRACTPseudomonas fluorescensand related plant root (“rhizosphere”)-associated species contribute to plant health by modulating defenses and facilitating nutrient uptake. To identify bacterial fitness determinants in the rhizosphere of the model plantArabidopsis thaliana, we performed a high-throughput transposon sequencing (Tn-Seq) screen using the biocontrol and growth-promoting strainPseudomonassp. WCS365. The screen, which was performed in parallel on wild-type and immunocompromisedArabidopsisplants, identified 231 genes that increased fitness in the rhizosphere of wild-type plants. A subset of these genes decreased fitness in the rhizosphere of immunocompromised plants. We hypothesized that these genes might be involved in avoiding plant defenses and verified 7Pseudomonassp. WCS365 candidate genes by generating clean deletions. We found that two of these deletion mutants, ΔmorA(encoding a putative diguanylate cyclase/phosphodiesterase) and ΔspuC(encoding a putrescine aminotransferase), formed enhanced biofilms and inhibited plant growth. We found that mutantsΔspuCandΔmorAinduced pattern-triggered immunity (PTI) as measured by induction of anArabidopsisPTI reporter andFLS2/BAK1-dependent inhibition of plant growth. We show that MorA acts as a phosphodiesterase to inhibit biofilm formation, suggesting a possible role in biofilm dispersal. We found that both putrescine and its precursor arginine promote biofilm formation that is enhanced in theΔspuCmutant, which cannot break down putrescine, suggesting that putrescine might serve as a signaling molecule in the rhizosphere. Collectively, this work identified novel bacterial factors required to evade plant defenses in the rhizosphere.IMPORTANCEWhile rhizosphere bacteria hold the potential to improve plant health and fitness, little is known about the bacterial genes required to evade host immunity. Using a model system consisting ofArabidopsisand a beneficialPseudomonassp. isolate, we identified bacterial genes required for both rhizosphere fitness and for evading host immune responses. This work advances our understanding of how evasion of host defenses contributes to survival in the rhizosphere.

scholarly journals Genome-Wide Analysis Reveals a RhlA-Dependent Modulation of Flagellar Genes in Pseudomonas Aeruginosa PAO1

2021 ◽  
Author(s):  
Michele Castro ◽  
Graciela Maria Dias ◽  
Tiago Salles ◽  
Núbia Cabral ◽  
Danielly Mariano ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Mutant Strain ◽  
Biofilm Formation ◽  
Dna Microarrays ◽  
Model Organism ◽  
Opportunistic Pathogen ◽  
Wild Type ◽  
Swarming Motility ◽  
Or Gene ◽  
Flagellar Genes

Abstract Background: Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role on biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids’ modulation of those group behaviors. Results: Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type P. aeruginosa PAO1 and a rhlA-mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the non-motile phenotype of the rhlA-minus strain. Swarming motility was restored with the addition of exogenous rhamnolipids obtained from the wild-type strain. Conclusions: Our results show significant evidence that rhamnolipids and/or their precursors, 3-(3-hydroxyalkanoyloxy) alkanoic acids, the major biosynthetic products of rhlABC pathway, seem to modulate gene expression in P. aeruginosa. Swarming motility assays support this hypothesis, since the non-motile rhlA-mutant strain had its swarming ability restored by the addition of exogenous rhamnolipids.

scholarly journals A genome-wide screen identifies genes in rhizosphere-associatedPseudomonasrequired to evade plant defenses

2018 ◽  
Author(s):  
Zhexian Liu ◽  
Polina Beskrovnaya ◽  
Ryan A. Melnyk ◽  
Sarzana S. Hossain ◽  
Sophie Khorasani ◽  
...  
Keyword(s):  
Plant Growth ◽  
Biofilm Formation ◽  
Plant Defenses ◽  
Plant Root ◽  
Plant Health ◽  
Wild Type ◽  
Host Immune Responses ◽  
A Genome ◽  
Dependent Inhibition ◽  
Bacterial Genes

AbstractPseudomonas fluorescensand related plant root- (“rhizosphere”) associated species contribute to plant health by modulating defenses and facilitating nutrient uptake. To identify bacterial fitness determinants in the rhizosphere of the model plantArabidopsis thaliana, we performed a Tn-Seq screen using the biocontrol and growth-promoting strainPseudomonassp. WCS365. The screen, which was performed in parallel on wild-type and an immunocompromisedArabidopsis, identified 231 genes that positively affect fitness in the rhizosphere of wild-type plants. A subset of these genes negatively affect fitness in the rhizosphere of immunocompromised plants. We postulated that these genes might be involved in avoiding plant defenses and verified 7Pseudomonassp. WCS365 candidate genes by generating clean deletions. We found that two of these deletion strains, ΔmorA(encodes a putative diguanylate cyclase/phosphodiesterase) and ΔspuC(encodes a putrescine aminotransferase) formed enhanced biofilms and inhibited plant growth. Inhibition of plant growth by ΔspuCand ΔmorAwas the result of pattern triggered immunity (PTI) as measured by induction of anArabidopsisPTI reporter andFLS2/BAK1-dependent inhibition of plant growth. We found that MorA acts as a phosphodiesterase to inhibit biofilm formation suggesting a possible role in biofilm dispersal. We found that both putrescine and its precursor arginine promote biofilm formation that is enhanced in the ΔspuCmutant, which cannot break down putrescine suggesting that putrescine might serve as a signaling molecule in the rhizosphere. Collectively, this work identified novel bacterial factors required to evade plant defenses in the rhizosphere.ImportanceWhile rhizosphere bacteria hold the potential to improve plant health and fitness, little is known about the bacterial genes required to evade host immunity. Using a model system consisting ofArabidopsisand a beneficialPseudomonassp. isolate, we identified bacterial genes required for both rhizosphere fitness and for evading host immune responses. This work advances our understanding of how evasion of host defenses contributes to survival in the rhizosphere.

scholarly journals The Variable Region of Pneumococcal Pathogenicity Island 1 Is Responsible for Unusually High Virulence of a Serotype 1 Isolate

2016 ◽  
Vol 84 (3) ◽  
pp. 822-832 ◽  
Author(s):  
Richard M. Harvey ◽  
Claudia Trappetti ◽  
Layla K. Mahdi ◽  
Hui Wang ◽  
Lauren J. McAllister ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Variable Region ◽  
Pathogenicity Island ◽  
Interleukin 10 ◽  
Wild Type ◽  
Altered Expression ◽  
Content Type ◽  
Genes Encoding ◽  
Serotype 1 ◽  
Phagocytic Killing

Streptococcus pneumoniaeis the leading infectious cause of death in children in the world. However, the mechanisms that drive the progression from asymptomatic colonization to disease are poorly understood. Two virulence-associated genomic accessory regions (ARs) were deleted in a highly virulent serotype 1 clinical isolate (strain 4496) and examined for their contribution to pathogenesis. Deletion of a prophage encoding a platelet-binding protein (PblB) resulted in reduced adherence, biofilm formation, reduced initial infection within the lungs, and a reduction in the number of circulating platelets in infected mice. However, the region's overall contribution to the survival of mice was not significant. In contrast, deletion of the variable region of pneumococcal pathogenicity island 1 (vPPI1) was also responsible for a reduction in adherence and biofilm formation but also reduced survival and invasion of the pleural cavity, blood, and lungs. While the 4496ΔPPI1 strain induced higher expression of the genes encoding interleukin-10 (IL-10) and CD11b in the lungs of challenged mice than the wild-type strain, very few other genes exhibited altered expression. Moreover, while the level of IL-10 protein was increased in the lungs of 4496ΔPPI1 mutant-infected mice compared to strain 4496-infected mice, the levels of gamma interferon (IFN-γ), CXCL10, CCL2, and CCL4 were not different in the two groups. However, the 4496ΔPPI1 mutant was found to be more susceptible than the wild type to phagocytic killing by a macrophage-like cell line. Therefore, our data suggest that vPPI1 may be a major contributing factor to the heightened virulence of certain serotype 1 strains, possibly by influencing resistance to phagocytic killing.

scholarly journals FimR and FimS: Biofilm Formation and Gene Expression in Porphyromonas gingivalis

2010 ◽  
Vol 192 (5) ◽  
pp. 1332-1343 ◽  
Author(s):  
Alvin Lo ◽  
Christine Seers ◽  
Stuart Dashper ◽  
Catherine Butler ◽  
Glenn Walker ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Dna Microarrays ◽  
Response Regulator ◽  
Strain Atcc ◽  
Wild Type ◽  
Signal Transduction System ◽  
Genes Encoding ◽  
Two Component Signal Transduction ◽  
Plaque Biofilm ◽  
Type Strains

ABSTRACT Porphyromonas gingivalis is a late-colonizing bacterium of the subgingival dental plaque biofilm associated with periodontitis. Two P. gingivalis genes, fimR and fimS, are predicted to encode a two-component signal transduction system comprising a response regulator (FimR) and a sensor histidine kinase (FimS). In this study, we show that fimS and fimR, although contiguous on the genome, are not part of an operon. We inactivated fimR and fimS in both the afimbriated strain W50 and the fimbriated strain ATCC 33277 and demonstrated that both mutants formed significantly less biofilm than their respective wild-type strains. Quantitative reverse transcription-real-time PCR showed that expression of fimbriation genes was reduced in both the fimS and fimR mutants of strain ATCC 33277. The mutations had no effect, in either strain, on the P. gingivalis growth rate or on the response to hydrogen peroxide or growth at pH 9, at 41°C, or at low hemin availability. Transcriptome analysis using DNA microarrays revealed that inactivation of fimS resulted in the differential expression of 10% of the P. gingivalis genome (>1.5-fold; P < 0.05). Notably genes encoding seven different transcriptional regulators, including the fimR gene and three extracytoplasmic sigma factor genes, were differentially expressed in the fimS mutant.

scholarly journals Role of MrkJ, a Phosphodiesterase, in Type 3 Fimbrial Expression and Biofilm Formation in Klebsiella pneumoniae

2010 ◽  
Vol 192 (15) ◽  
pp. 3944-3950 ◽  
Author(s):  
Jeremiah G. Johnson ◽  
Steven Clegg
Keyword(s):  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Opportunistic Pathogen ◽  
Surface Expression ◽  
Reverse Transcription Pcr ◽  
Genes Encoding ◽  
Bacterial Genes ◽  
Type 3

ABSTRACT Klebsiella pneumoniae is an opportunistic pathogen that has been shown to adhere to human extracellular matrices using the type 3 fimbriae. Introduction of plasmids carrying genes known to alter intracellular cyclic-di-GMP pools in Vibrio parahaemolyticus revealed that these genes also altered type 3 fimbrial surface expression in K. pneumoniae. Immediately adjacent to the type 3 fimbrial gene cluster is a gene, mrkJ, that is related to a family of bacterial genes encoding phosphodiesterases. We identify here a role for MrkJ, a functional phosphodiesterase exhibiting homology to EAL domain-containing proteins, in controlling type 3 fimbria production and biofilm formation in K. pneumoniae. Deletion of mrkJ resulted in an increase in type 3 fimbria production and biofilm formation as a result of the accumulation of intracellular cyclic-di-GMP. This gene was shown to encode a functional phosphodiesterase via restoration of motility in a V. parahaemolyticus strain previously shown to accumulate cyclic-di-GMP and in vitro using phosphodiesterase activity assays. The effect of the mrkJ mutation on type 3 fimbrial expression was shown to be at the level of mrkA gene transcription by using quantitative reverse transcription-PCR. These results reveal a previously unknown role for cyclic-di-GMP in type 3 fimbrial production.

scholarly journals Characterization of Vibrio cholerae RyhB: the RyhB Regulon and Role of ryhB in Biofilm Formation

2005 ◽  
Vol 73 (9) ◽  
pp. 5706-5719 ◽  
Author(s):  
Alexandra R. Mey ◽  
Stephanie A. Craig ◽  
Shelley M. Payne
Keyword(s):  
Vibrio Cholerae ◽  
Biofilm Formation ◽  
Iron Homeostasis ◽  
Tricarboxylic Acid Cycle ◽  
Transport Systems ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Iron Deficient ◽  
Genes Encoding

ABSTRACT Vibrio cholerae encodes a small RNA with homology to Escherichia coli RyhB. Like E. coli ryhB, V. cholerae ryhB is negatively regulated by iron and Fur and is required for repression of genes encoding the superoxide dismutase SodB and multiple tricarboxylic acid cycle enzymes. However, V. cholerae RyhB is considerably longer (>200 nucleotides) than the E. coli RNA (90 nucleotides), and it regulates the expression of a variety of genes that are not known to be regulated by RyhB in E. coli, including genes involved in motility, chemotaxis, and biofilm formation. A mutant with a deletion in ryhB had reduced chemotactic motility in low-iron medium and was unable to form wild-type biofilms. The defect in biofilm formation was suppressed by growing the mutant in the presence of excess iron or succinate. The wild-type strain showed reduced biofilm formation in iron-deficient medium, further supporting a role for iron in normal biofilm formation. The ryhB mutant was not defective for colonization in a mouse model and appeared to be at a slight advantage when competing with the wild-type parental strain. Other genes whose expression was influenced by RyhB included those encoding the outer membrane porins OmpT and OmpU, several iron transport systems, and proteins containing heme or iron-sulfur clusters. These data indicate that V. cholerae RyhB has diverse functions, ranging from iron homeostasis to the regulation of biofilm formation.

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