scholarly journals Effects of Sequential Campylobacter jejuni 81-176 Lipooligosaccharide Core Truncations on Biofilm Formation, Stress Survival, and Pathogenesis

2010 ◽  
Vol 192 (8) ◽  
pp. 2182-2192 ◽  
Author(s):  
Mizue Naito ◽  
Emilisa Frirdich ◽  
Joshua A. Fields ◽  
Mark Pryjma ◽  
Jianjun Li ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Biofilm Formation ◽  
Cell Envelope ◽  
Polymyxin B ◽  
Outer Core ◽  
Survival Strategies ◽  
Calcofluor White ◽  
Stress Survival ◽  
Insight Into

ABSTRACTCampylobacter jejuniis a highly prevalent human pathogen for which pathogenic and stress survival strategies remain relatively poorly understood. We previously found that aC. jejunistrain 81-176 mutant defective for key virulence and stress survival attributes was also hyper-biofilm and hyperreactive to the UV fluorescent dye calcofluor white (CFW). We hypothesized that screening for CFW hyperreactive mutants would identify additional genes required forC. jejunipathogenesis properties. Surprisingly, two such mutants harbored lesions in lipooligosaccharide (LOS) genes (waaFandlgtF), indicating a complete loss of the LOS outer core region. We utilized this as an opportunity to explore the role of each LOS core-specific moiety in the pathogenesis and stress survival of this strain and thus also constructed ΔgalTand ΔcstIImutants with more minor LOS truncations. Interestingly, we found that mutants lacking the LOS outer core (ΔwaaFand ΔlgtFbut not ΔgalTor ΔcstIImutants) exhibited enhanced biofilm formation. The presence of the complete outer core was also necessary for resistance to complement-mediated killing. In contrast, any LOS truncation, even that of the terminal sialic acid (ΔcstII), resulted in diminished resistance to polymyxin B. The cathelicidin LL-37 was found to be active againstC. jejuni, with the LOS mutants exhibiting modest but tiled alterations in LL-37 sensitivity. The ΔwaaFmutant but not the other LOS mutant strains also exhibited a defect in intraepithelial cell survival, an aspect ofC. jejunipathogenesis that has only recently begun to be clarified. Finally, using a mouse competition model, we now provide the first direct evidence for the importance of theC. jejuniLOS in host colonization. Collectively, this study has uncovered novel roles for theC. jejuniLOS, highlights the dynamic nature of theC. jejunicell envelope, and provides insight into the contribution of specific LOS core moieties to stress survival and pathogenesis.

scholarly journals Histone-Like Nucleoid Structuring Protein Modulates the Fitness of tet(X4)-Bearing IncX1 Plasmids in Gram-Negative Bacteria

2021 ◽  
Vol 12 ◽  
Author(s):  
Wenhui Cai ◽  
Feifei Tang ◽  
Lijie Jiang ◽  
Ruichao Li ◽  
Zhiqiang Wang ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Antibiotic Resistance Genes ◽  
Inhibitory Effect ◽  
Fitness Cost ◽  
Relative Fitness ◽  
Gram Negative Bacteria ◽  
Underlying Mechanisms ◽  
Biological Environment ◽  
Insight Into

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) poses a challenging threat to public health. Based on the analysis of tet(X4)-positive plasmids in the NCBI database, we found that the IncX1-type plasmid is one of the most common vectors for spreading tet(X4) gene, but the mechanisms by which these plasmids adapt to host bacteria and maintain the persistence of antibiotic resistance genes (ARGs) remain unclear. Herein, we investigated the underlying mechanisms of how host bacteria modulate the fitness cost of IncX1 plasmids carrying tet(X4) gene. Interestingly, we found that the tet(X4)-bearing IncX1 plasmids encoding H-NS protein imposed low or no fitness cost in Escherichia coli and Klebsiella pneumoniae; instead, they partially promoted the virulence and biofilm formation in host bacteria. Regression analysis revealed that the expression of hns gene in plasmids was positively linked to the relative fitness of host bacteria. Furthermore, when pCE2::hns was introduced, the fitness of tet(X4)-positive IncX1 plasmid pRF55-1 without hns gene was significantly improved, indicating that hns mediates the improvement of fitness. Finally, we showed that the expression of hns gene is negatively correlated with the expression of tet(X4) gene, suggesting that the regulatory effect of H-NS on adaptability may be attributed to its inhibitory effect on the expression of ARGs. Together, our findings suggest the important role of plasmid-encoded H-NS protein in modulating the fitness of tet(X4)-bearing IncX1 plasmids, which shed new insight into the dissemination of tet(X4) gene in a biological environment.

scholarly journals Analysis of the Roles of FlgP and FlgQ in Flagellar Motility of Campylobacter jejuni

2006 ◽  
Vol 189 (1) ◽  
pp. 179-186 ◽  
Author(s):  
Shawn M. Sommerlad ◽  
David R. Hendrixson
Keyword(s):  
Outer Membrane ◽  
Campylobacter Jejuni ◽  
Important Determinant ◽  
Flagellar Motility ◽  
Transposon Insertion ◽  
Flagellar Biosynthesis ◽  
Transcriptional Regulatory ◽  
Regulatory Cascades ◽  
Insight Into

ABSTRACT Flagellar motility is an important determinant of Campylobacter jejuni that is required for promoting interactions with various hosts to promote gastroenteritis in humans or commensal colonization of many animals. In a previous study, we identified a nonmotile mutant of C. jejuni 81-176 with a transposon insertion in Cj1026c, but verification of the role of the encoded protein in motility was not determined. In this study, we have determined that Cj1026c and the gene immediately downstream, Cj1025c (here annotated as flgP and flgQ, respectively), are both required for motility of C. jejuni but not for flagellar biosynthesis. FlgP and FlgQ are not components of the transcriptional regulatory cascades to activate σ28- or σ54-dependent expression of flagellar genes. In addition, expression of flgP and flgQ is not largely dependent on σ28 or σ54. Immunblot analyses revealed that the majority of FlgP in C. jejuni is associated with the outer membrane. However, in the absence of FlgQ, the amounts of FlgP in the outer membrane of C. jejuni are greatly reduced, suggesting that FlgQ may be required for localization or stability of FlgP at this location. This study provides insight into features of FlgP and FlgQ, two proteins with previously undefined functions that are required for the larger, multicomponent flagellar system of C. jejuni that is necessary for motility.

scholarly journals Structural insight into the role of Streptococcus parasanguinis Fap1 within oral biofilm formation

2012 ◽  
Vol 417 (1) ◽  
pp. 421-426 ◽  
Author(s):  
James A. Garnett ◽  
Peter J. Simpson ◽  
Jonathan Taylor ◽  
Stefi V. Benjamin ◽  
Camille Tagliaferri ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Oral Biofilm ◽  
Structural Insight ◽  
Insight Into ◽  
Streptococcus Parasanguinis

scholarly journals Campylobacter jejuni Biofilms Up-Regulated in the Absence of the Stringent Response Utilize a Calcofluor White-Reactive Polysaccharide

2007 ◽  
Vol 190 (3) ◽  
pp. 1097-1107 ◽  
Author(s):  
Meghan K. McLennan ◽  
Danielle D. Ringoir ◽  
Emilisa Frirdich ◽  
Sarah L. Svensson ◽  
Derek H. Wells ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Stringent Response ◽  
Wild Type ◽  
Calcofluor White ◽  
Targeted Deletion ◽  
Reactive Polymer ◽  
Surface Polysaccharides

ABSTRACT The enteric pathogen Campylobacter jejuni is a highly prevalent yet fastidious bacterium. Biofilms and surface polysaccharides participate in stress survival, transmission, and virulence in C. jejuni; thus, the identification and characterization of novel genes involved in each process have important implications for pathogenesis. We found that C. jejuni reacts with calcofluor white (CFW), indicating the presence of surface polysaccharides harboring β1-3 and/or β1-4 linkages. CFW reactivity increased with extended growth, under 42°C anaerobic conditions, and in a ΔspoT mutant defective for the stringent response (SR). Conversely, two newly isolated dim mutants exhibited diminished CFW reactivity as well as growth and serum sensitivity differences from the wild type. Genetic, biochemical, and nuclear magnetic resonance analyses suggested that differences in CFW reactivity between wild-type and ΔspoT and dim mutant strains were independent of well-characterized lipooligosaccharides, capsular polysaccharides, and N-linked polysaccharides. Targeted deletion of carB downstream of the dim13 mutation also resulted in CFW hyporeactivity, implicating a possible role for carbamoylphosphate synthase in the biosynthesis of this polysaccharide. Correlations between biofilm formation and production of the CFW-reactive polymer were demonstrated by crystal violet staining, scanning electron microscopy, and confocal microscopy, with the C. jejuni ΔspoT mutant being the first SR mutant in any bacterial species identified as up-regulating biofilms. Together, these results provide new insight into genes and processes important for biofilm formation and polysaccharide production in C. jejuni.

scholarly journals Alternative Sigma Factor RpoE Is Important for Vibrio parahaemolyticus Cell Envelope Stress Response and Intestinal Colonization

2014 ◽  
Vol 82 (9) ◽  
pp. 3667-3677 ◽  
Author(s):  
Brandy Haines-Menges ◽  
W. Brian Whitaker ◽  
E. Fidelma Boyd
Keyword(s):  
Stress Response ◽  
Vibrio Parahaemolyticus ◽  
Response Regulator ◽  
Cell Envelope ◽  
Polymyxin B ◽  
Content Type ◽  
Alternative Sigma Factors ◽  
Envelope Stress

ABSTRACTVibrio parahaemolyticusis a halophile that inhabits brackish waters and a wide range of hosts, including crustaceans, fish, mollusks, and humans. In humans, it is the leading cause of bacterial seafood-borne gastroenteritis. The focus of this work was to determine the role of alternative sigma factors in the stress response ofV. parahaemolyticusRIMD2210633, an O3:K6 pandemic isolate. Bioinformatics identified five putative extracytoplasmic function (ECF) family of alternative sigma factors: VP0055, VP2210, VP2358, VP2578, and VPA1690. ECF factors typically respond to cell wall/cell envelope stress, iron levels, and the oxidation state of the cell. We have demonstrated here that one such sigma factor, VP2578, a homologue of RpoE fromEscherichia coli, is important for survival under a number of cell envelope stress conditions and in gastrointestinal colonization of a streptomycin-treated adult mouse. In this study, we determined that anrpoEdeletion mutant strain BHM2578 compared to the wild type (WT) was significantly more sensitive to polymyxin B, ethanol, and high-temperature stresses. We demonstrated that inin vivocompetition assays between therpoEmutant and the WT marked with the β-galactosidase genelacZ(WBWlacZ), the mutant strain was defective in colonization compared to the WT. In contrast, deletion of therpoSstress response regulator did not affectin vivosurvival. In addition, we examined the role of the outer membrane protein, OmpU, which inV. choleraeis proposed to be the sole activator of RpoE. We found that anompUdeletion mutant was sensitive to bile salt stress but resistant to polymyxin B stress, indicating OmpU is not essential for the cell envelope stress responses or RpoE function. Overall, these data demonstrate that RpoE is a key cell envelope stress response regulator and, similar toE. coli, RpoE may have several factors that stimulate its function.

scholarly journals Mutagenic strategies against luxS gene affect the early stage of biofilm formation of Campylobacter jejuni

2021 ◽  
Author(s):  
Martin Tereň ◽  
Ekaterina Shagieva ◽  
Lucie Vondrakova ◽  
Jitka Viktorova ◽  
Viviana Svarcova ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Biofilm Formation ◽  
Early Stage ◽  
Wild Type ◽  
Genetic Determinants ◽  
Autoinducer 2 ◽  
Naturally Occurring ◽  
Mutant Strains ◽  
The Impact

Abstract Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81–176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated (⸬luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.

scholarly journals Role of Ring6 in the function of the E. coli MCE protein LetB

2021 ◽  
Author(s):  
Casey Vieni ◽  
Nicolas Coudray ◽  
Georgia L Isom ◽  
Gira Bhabha ◽  
Damian Charles Ekiert
Keyword(s):  
Cell Envelope ◽  
Primary Sequence ◽  
Functional Roles ◽  
E Coli ◽  
Outer Membranes ◽  
Gating Mechanism ◽  
Pore Lining ◽  
Insight Into ◽  
Modular Nature

LetB is a tunnel-forming protein found in the cell envelope of some double-membraned bacteria, and is thought to be important for the transport of lipids between the inner and outer membranes. In Escherichia coli the LetB tunnel is formed from a stack of seven rings (Ring1 - Ring7), in which each ring is composed of a homo-hexameric assembly of MCE domains. The primary sequence of each MCE domain of the LetB protein is substantially divergent from the others, making each MCE ring unique in nature. The role of each MCE domain and how it contributes to the function of LetB is not well understood. Here we probed the importance of each MCE ring for the function of LetB, using a combination of bacterial growth assays and cryo-EM. Surprisingly, we find that ΔRing3 and ΔRing6 mutants, in which Ring3 and Ring6 have been deleted, confer increased resistance to membrane perturbing agents. Specific mutations in the pore-lining loops of Ring6 similarly confer increased resistance. A cryo-EM structure of the ΔRing6 mutant shows that despite the absence of Ring6, which leads to a shorter assembly, the overall architecture is maintained, highlighting the modular nature of MCE proteins. Previous work has shown that Ring6 is dynamic and in its closed state, may restrict the passage of substrate through the tunnel. Our work suggests that removal of Ring6 may relieve this restriction. The deletion of Ring6 combined with mutations in the pore-lining loops leads to a model for the tunnel gating mechanism of LetB. Together, these results provide insight into the functional roles of individual MCE domains and pore-lining loops in the LetB protein.

scholarly journals YodL and YisK Possess Shape-Modifying Activities That Are Suppressed by Mutations in Bacillus subtilismreBandmbl

2016 ◽  
Vol 198 (15) ◽  
pp. 2074-2088 ◽  
Author(s):  
Yi Duan ◽  
Anthony M. Sperber ◽  
Jennifer K. Herman
Keyword(s):  
Cell Shape ◽  
Cell Envelope ◽  
Critical Role ◽  
Binding Pocket ◽  
Cell Width ◽  
Content Type ◽  
Structural Rigidity ◽  
Sporulation Efficiency ◽  
Insight Into

ABSTRACTMany bacteria utilize actin-like proteins to direct peptidoglycan (PG) synthesis. MreB and MreB-like proteins are thought to act as scaffolds, guiding the localization and activity of key PG-synthesizing proteins during cell elongation. Despite their critical role in viability and cell shape maintenance, very little is known about how the activity of MreB family proteins is regulated. Using aBacillus subtilismisexpression screen, we identified two genes,yodLandyisK, that when misexpressed lead to loss of cell width control and cell lysis. Expression analysis suggested thatyodLandyisKare previously uncharacterized Spo0A-regulated genes, and consistent with these observations, a ΔyodLΔyisKmutant exhibited reduced sporulation efficiency. Suppressors resistant to YodL's killing activity occurred primarily inmreBmutants and resulted in amino acid substitutions at the interface between MreB and the highly conserved morphogenic protein RodZ, whereas suppressors resistant to YisK occurred primarily inmblmutants and mapped to Mbl's predicted ATP-binding pocket. YodL's shape-altering activity appears to require MreB, as a ΔmreBmutant was resistant to the effects of YodL but not YisK. Similarly, YisK appears to require Mbl, as a Δmblmutant was resistant to the cell-widening effects of YisK but not of YodL. Collectively, our results suggest that YodL and YisK likely modulate MreB and Mbl activity, possibly during the early stages of sporulation.IMPORTANCEThe peptidoglycan (PG) component of the cell envelope confers structural rigidity to bacteria and protects them from osmotic pressure. MreB and MreB-like proteins are thought to act as scaffolds for PG synthesis and are essential in bacteria exhibiting nonpolar growth. Despite the critical role of MreB-like proteins, we lack mechanistic insight into how their activities are regulated. Here, we describe the discovery of twoB. subtilisproteins, YodL and YisK, which modulate MreB and Mbl activities. Our data suggest that YodL specifically targets MreB, whereas YisK targets Mbl. The apparent specificities with which YodL and YisK are able to differentially target MreB and Mbl make them potentially powerful tools for probing the mechanics of cytoskeletal function in bacteria.

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