Pilus Gene Research Articles

Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster.

Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.

In vitro characterization of biofilms formed by Kingella kingae.

The Gram-negative bacterium Kingella kingae is part of the normal oropharyngeal mucosal flora of children <4years old. K. kingae can enter the submucosa and cause infections of the skeletal system in children, including septic arthritis and osteomyelitis. The organism is also associated with infective endocarditis in children and adults. Although biofilm formation has been coupled with pharyngeal colonization, osteoarticular infections, and infective endocarditis, no studies have investigated biofilm formation in K.kingae. In this study we measured biofilm formation by 79 K.kingae clinical isolates using a 96-well microtiter plate crystal violet binding assay. We found that 37 of 79 strains (47%) formed biofilms. All strains that formed biofilms produced corroding colonies on agar. Biofilm formation was inhibited by proteinase K and DNase I. DNase I also caused the detachment of pre-formed K.kingae biofilm colonies. A mutant strain carrying a deletion of the pilus gene cluster pilA1pilA2fimB did not produce corroding colonies on agar, autoaggregate in broth, or form biofilms. Biofilm forming strains have higher levels of pilA1 expression. The extracellular components of biofilms contained 490μgcm-2 of protein, 0.68μgcm-2 of DNA, and 0.4μgcm-2 of total carbohydrates. We concluded that biofilm formation is common among K.kingae clinical isolates, and that biofilm formation is dependent on the production of proteinaceous pili and extracellular DNA. Biofilm development may have relevance to the colonization, transmission, and pathogenesis of this bacterium. Extracellular DNA production by K.kingae may facilitate horizontal gene transfer within the oral microbial community.

Heat stress-induced activation of a Trichoderma harzianum PIL superfamily gene

Various plasma membrane compartments in fungal cell wall are involved in a wide range of cellular functions. Eisosome protein Pil1 (phosphorylation is inhibited by long chain bases) in yeast cells are known to be involved genetic pathway that controls the cellular responses to heat stress. The PIL gene superfamily is well conserved among different organisms. In this study, a full-length ThPIL1 gene that encodes for a 357 amino acid protein was cloned from Trichoderma harzianum T35 strain. A comparative analysis of ThPIL1 gene sequence with other PIL1 genes of different organisms and Trichoderma spp. shows degree of sequence identity ranging from 78 to 95%. Southern blot analysis showed that there existed only one copy of the ThPIL1 gene of in genome of T. harzianum T35, but alternative splicing of ThPIL1 generates two transcript isoforms, ThPIL1-α and β. Spliced transcript ThPIL1-β retains an unspliced first intron that carries a premature termination codon. The expression characteristics of the both isoforms of ThPIL1 gene in the mycelium grown at different temperatures were investigated using quantitative real-time PCR (qRT-PCR). The results showed that they are differentially expressed in mycelia exposed to different temperatures, the major product being ThPIL1-α. The transcripts of ThPIL1-β accumulated markedly when the Trichoderma mycelium was subjected to heat (40°C and 45°C). The expression level of the ThPIL1-α was highest in treatment when mycelium was grown at 45°C for 2h, and was approximately 4-fold higher than at 45°C for 1h. The highest expression level of ThPIL1-β was detected at the same temperature, and very negligible expression was found at lower temperature. These results provide new insight into roles in the expression regulation of fungal eisosome genes and suggest that ThPIL1 gene may play a role in growth of T. harzianum T35 under the heat stress response to environmental stresses.

A genomic island in Vibrio cholerae with VPI-1 site-specific recombination characteristics contains CRISPR-Cas and type VI secretion modules.

Cholera is a devastating diarrhoeal disease caused by certain strains of serogroup O1/O139 Vibrio cholerae. Mobile genetic elements such as genomic islands (GIs) have been pivotal in the evolution of O1/O139 V. cholerae. Perhaps the most important GI involved in cholera disease is the V. cholerae pathogenicity island 1 (VPI-1). This GI contains the toxin-coregulated pilus (TCP) gene cluster that is necessary for colonization of the human intestine as well as being the receptor for infection by the cholera-toxin bearing CTX phage. In this study, we report a GI (designated GIVchS12) from a non-O1/O139 strain of V. cholerae that is present in the same chromosomal location as VPI-1, contains an integrase gene with 94% nucleotide and 100% protein identity to the VPI-1 integrase, and attachment (att) sites 100% identical to those found in VPI-1. However, instead of TCP and the other accessory genes present in VPI-1, GIVchS12 contains a CRISPR-Cas element and a type VI secretion system (T6SS). GIs similar to GIVchS12 were identified in other V. cholerae genomes, also containing CRISPR-Cas elements and/or T6SS’s. This study highlights the diversity of GIs circulating in natural V. cholerae populations and identifies GIs with VPI-1 recombination characteristics as a propagator of CRISPR-Cas and T6SS modules.

Toxigenic Vibrio cholerae O1 in vegetables and fish raised in wastewater irrigated fields and stabilization ponds during a non-cholera outbreak period in Morogoro, Tanzania: an environmental health study.

BackgroundCholera, one of the world’s deadliest infectious diseases, remains rampant and frequent in Tanzania and thus hinders existing control measures. The present study was undertaken to evaluate the occurrence of toxigenic Vibrio cholerae O1 in wastewater, fish and vegetables during a non-outbreak period in Morogoro, Tanzania.MethodsFrom October 2014 to February 2015, 60 wastewater samples, 60 fish samples from sewage stabilization ponds and 60 wastewater irrigated vegetable samples were collected. Samples were cultured for identification of V. cholerae using conventional bacteriological methods. Isolates were confirmed as V. cholerae by detection of the outer membrane protein gene (ompW) using polymerase chain reaction (PCR). Isolates were further tested for antibiotic susceptibility and presence of virulence genes including, cholera enterotoxin gene (ctx), the toxin co-regulated pilus gene (tcpA) and the haemolysin gene (hlyA).ResultsThe prevalence of V. cholerae in wastewater, vegetables and fish was 36.7, 21.7 and 23.3 %, respectively. Two isolates from fish gills were V. cholerae O1 and tested positive for ctx and tcpA. One of these contained in addition the hlyA gene while five isolates from fish intestines tested positive for tcpA. All V. cholerae isolates were resistant to ampicillin, amoxicillin and some to tetracycline, but sensitive to gentamicin, chloramphenicol, and ciprofloxacin.ConclusionsOur results show that toxigenic and drug-resistant V. cholerae O1 species are present and persist in aquatic environments during a non-cholera outbreak period. This is of public health importance and shows that such environments may be important as reservoirs and in the transmission of V. cholerae O1.

Diversity of plasmids and Tn1546-type transposons among VanA Enterococcus faecium in Poland

The objective of this study was to investigate the antimicrobial resistance, Tn1546 transposon variability and plasmid diversity among Polish vancomycin-resistant Enterococcus faecium (VREfm) isolates of VanA phenotype in the context of their clonal structure. Two hundred sixteen clinical VREfm isolates collected between 1997 and 2010 were studied by antimicrobial susceptibility testing, MLST, MLVA and detection of IS16, espEfm, pilA, intA and plasmid-specific genes by PCR. Tn1546 structure was revealed by overlapping PCR and sequencing. Selected isolates were subjected to PFGE-S1 and Southern hybridization analyses. The vast majority of the isolates (95.8 %) belonged to lineages 17/18 (during the whole study period 1997–2010) and 78 (mostly in 2006–2010) of hospital-adapted meroclone of E. faecium. All isolates displayed a multi-drug resistance phenotype. Twenty-eight Tn1546 types (including 26 novel ones) were associated with eight different ISs (IS1216, IS1251, ISEfa4, ISEfa5, ISEfm2, ISEf1, IS3-like, ISEfm1-like). The vanA-determinant was typically located on plasmids, which most commonly carried rep2pRE25, rep17pRUM, rep18pEF418, rep1pIP501, ω-ε-ζ and axe-txe genes. VanA isolates from 1997–2005 to 2006–2010 differed in clonal composition, prevalence of gentamicin- and tetracycline-resistance and plasmidome. Our analysis revealed high complexity of Tn1546-type transposons and vanA-plasmids, and suggested that diverse genetic events, such as conjugation transfer, recombination, chromosomal integration and DNA mutations shaped the structure of these elements among Polish VREfm.

Horizontal Transfer of the Salmonella enterica Serovar Infantis Resistance and Virulence Plasmid pESI to the Gut Microbiota of Warm-Blooded Hosts.

ABSTRACTSalmonella enterica serovar Infantis is one of the prevalent salmonellae worldwide. Recently, we showed that the emergence of S. Infantis in Israel was facilitated by the acquisition of a unique megaplasmid (pESI) conferring multidrug resistance and increased virulence phenotypes. Here we elucidate the ecology, transmission properties, and regulation of pESI. We show that despite its large size (~280 kb), pESI does not impose a significant metabolic burden in vitro and that it has been recently fixed in the domestic S. Infantis population. pESI conjugation and the transcription of its pilus (pil) genes are inhibited at the ambient temperature (27°C) and by ≥1% bile but increased under temperatures of 37 to 41°C, oxidative stress, moderate osmolarity, and the microaerobic conditions characterizing the intestinal environment of warm-blooded animals. The pESI-encoded protein TraB and the oxygen homeostasis regulator Fnr were identified as transcriptional regulators of pESI conjugation. Using the mouse model, we show that following S. Infantis infection, pESI can be horizontally transferred to the gut microbiota, including to commensal Escherichia coli strains. Possible transfer, but not persistence, of pESI was also observed into Gram-positive mouse microbiota species, especially Lactobacillus reuteri. Moreover, pESI was demonstrated to further disseminate from gut microbiota to S. enterica serovar Typhimurium, in the context of gastrointestinal infection. These findings exhibit the ability of a selfish clinically relevant megaplasmid to distribute to and from the microbiota and suggest an overlooked role of the microbiota as a reservoir of mobile genetic elements and intermediator in the spread of resistance and virulence genes between commensals and pathogenic bacteria.

Loop structures in the 5' untranslated region and antisense RNA mediate pilE gene expression in Neisseria gonorrhoeae.

Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem-loop structures with favourable free energies within the 5' untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem-loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5' untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.

Analysis of the contribution of MTP and the predicted Flp pilus genes to Mycobacterium tuberculosis pathogenesis.

Mycobacterium tuberculosis (Mtb) is one of the world's most successful pathogens. Millions of new cases of tuberculosis occur each year, emphasizing the need for better methods of treatment. The design of novel therapeutics is dependent on our understanding of factors that are essential for pathogenesis. Many bacterial pathogens use pili and other adhesins to mediate pathogenesis. The recently identified Mycobacterium tuberculosis pilus (MTP) and the hypothetical, widely conserved Flp pilus have been speculated to be important for Mtb virulence based on in vitro studies and homology to other pili, respectively. However, the roles for these pili during infection have yet to be tested. We addressed this gap in knowledge and found that neither MTP nor the hypothetical Flp pilus is required for Mtb survival in mouse models of infection, although MTP can contribute to biofilm formation and subsequent isoniazid tolerance. However, differences in mtp expression did affect lesion architecture in infected lungs. Deletion of mtp did not correlate with loss of cell-associated extracellular structures as visualized by transmission electron microscopy in Mtb Erdman and HN878 strains, suggesting that the phenotypes of the mtp mutants were not due to defects in production of extracellular structures. These findings highlight the importance of testing the virulence of adhesion mutants in animal models to assess the contribution of the adhesin to infection. This study also underscores the need for further investigation into additional strategies that Mtb may use to adhere to its host so that we may understand how this pathogen invades, colonizes and disseminates.

Positive Selection Pressure Drives Variation on the Surface-Exposed Variable Proteins of the Pathogenic Neisseria.

Pathogenic species of Neisseria utilize variable outer membrane proteins to facilitate infection and proliferation within the human host. However, the mechanisms behind the evolution of these variable alleles remain largely unknown due to analysis of previously limited datasets. In this study, we have expanded upon the previous analyses to substantially increase the number of analyzed sequences by including multiple diverse strains, from various geographic locations, to determine whether positive selective pressure is exerted on the evolution of these variable genes. Although Neisseria are naturally competent, this analysis indicates that only intrastrain horizontal gene transfer among the pathogenic Neisseria principally account for these genes exhibiting linkage equilibrium which drives the polymorphisms evidenced within these alleles. As the majority of polymorphisms occur across species, the divergence of these variable genes is dependent upon the species and is independent of geographical location, disease severity, or serogroup. Tests of neutrality were able to detect strong selection pressures acting upon both the opa and pil gene families, and were able to locate the majority of these sites within the exposed variable regions of the encoded proteins. Evidence of positive selection acting upon the hypervariable domains of Opa contradicts previous beliefs and provides evidence for selection of receptor binding. As the pathogenic Neisseria reside exclusively within the human host, the strong selection pressures acting upon both the opa and pil gene families provide support for host immune system pressure driving sequence polymorphisms within these variable genes.

Epistatic interactions between ancestral genotype and beneficial mutations shape evolvability in Pseudomonas aeruginosa.

The idea that interactions between mutations influence adaptation by driving populations to low and high fitness peaks on adaptive landscapes is deeply ingrained in evolutionary theory. Here, we investigate the impact of epistasis on evolvability by challenging populations of two Pseudomonas aeruginosa clones bearing different initial mutations (in rpoB conferring rifampicin resistance, and the type IV pili gene network) to adaptation to a medium containing l-serine as the sole carbon source. Despite being initially indistinguishable in fitness, populations founded by the two ancestral genotypes reached different fitness following 300 generations of evolution. Genome sequencing revealed that the difference could not be explained by acquiring mutations in different targets of selection; the majority of clones from both ancestors converged on one of the following two strategies: (1) acquiring mutations in either PA2449 (gcsR, an l-serine-metabolism RpoN enhancer binding protein) or (2) protease genes. Additionally, populations from both ancestors converged on loss-of-function mutations in the type IV pili gene network, either due to ancestral or acquired mutations. No compensatory or reversion mutations were observed in RNA polymerase (RNAP) genes, in spite of the large fitness costs typically associated with mutations in rpoB. Although current theory points to sign epistasis as the dominant constraint on evolvability, these results suggest that the role of magnitude epistasis in constraining evolvability may be underappreciated. The contribution of magnitude epistasis is likely to be greatest under the biologically relevant mutation supply rates that make back mutations probabilistically unlikely.

Truncated type IV pilin PilA(108) activates the intramembrane protease AlgW to cleave MucA and PilA(108) itself in vitro.

For alginate production in Pseudomonas aeruginosa, the intramembrane protease AlgW must be activated to cleave the periplasmic domain of anti-sigma factor MucA for release of the sequestered ECF sigma factor AlgU. Previously, we reported that three tandem point mutations in the pilA gene, resulting in a truncated type IV pilin termed PilA(108) with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF), induced mucoidy in strain PAO579. In this study, we purified PilA(108) protein and synthesized a peptide 'SGAGDITFTF' corresponding to C-terminus of PilA(108) and found they both caused the degradation of MucA by AlgW. Interestingly, AlgW could also cleave PilA(108) between alanine(62) and glycine(63) residues. Overexpression of the recombinant FTF motif-bearing MucE protein, originally a small periplasmic polypeptide with the C-terminal motif WVF, could induce mucoid conversion in the PAO1 strain. In all, our results provided a model of activation of AlgW by another protein ending with proper motifs. Our data suggest that in addition to MucA cleavage, AlgW may cleave other substrates.

The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation.

The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner.

ABSTRACTThe opportunistic pathogen Acinetobacter baumannii is able to persist in the environment and is often multidrug resistant (MDR), causing difficulties in the treatment of infections. Here, we show that the two-component system AdeRS, which regulates the production of the AdeABC multidrug resistance efflux pump, is required for the formation of a protective biofilm in an ex vivo porcine mucosal model, which mimics a natural infection of the human epithelium. Interestingly, deletion of adeB impacted only on the ability of strain AYE to form a biofilm on plastic and only on the virulence of strain Singapore 1 for Galleria mellonella. RNA-Seq revealed that loss of AdeRS or AdeB significantly altered the transcriptional landscape, resulting in the changed expression of many genes, notably those associated with antimicrobial resistance and virulence interactions. For example, A. baumannii lacking AdeRS displayed decreased expression of adeABC, pil genes, com genes, and a pgaC-like gene, whereas loss of AdeB resulted in increased expression of pil and com genes and decreased expression of ferric acinetobactin transport system genes. These data define the scope of AdeRS-mediated regulation, show that changes in the production of AdeABC mediate important phenotypes controlled by AdeRS, and suggest that AdeABC is a viable target for antimicrobial drug and antibiofilm discovery.

The ATP-dependent RNA helicase HrpB plays an important role in motility and biofilm formation in Xanthomonas citri subsp. citri.

BackgroundRNA helicases are enzymes that catalyze the separation of double-stranded RNA (dsRNA) using the free energy of ATP binding and hydrolysis. DEAD/DEAH families participate in many different aspects of RNA metabolism, including RNA synthesis, RNA folding, RNA-RNA interactions, RNA localization and RNA degradation. Several important bacterial DEAD/DEAH-box RNA helicases have been extensively studied. In this study, we characterize the ATP-dependent RNA helicase encoded by the hrpB (XAC0293) gene using deletion and genetic complementation assays. We provide insights into the function of the hrpB gene in Xanthomonas citri subsp. citri by investigating the roles of hrpB in biofilm formation on abiotic surfaces and host leaves, cell motility, host virulence of the citrus canker bacterium and growth in planta.ResultsThe hrpB gene is highly conserved in the sequenced strains of Xanthomonas. Mutation of the hrpB gene (∆hrpB) resulted in a significant reduction in biofilms on abiotic surfaces and host leaves. ∆hrpB also exhibited increased cell dispersion on solid medium plates. ∆hrpB showed reduced adhesion on biotic and abiotic surfaces and delayed development in disease symptoms when sprayed on susceptible citrus leaves. Quantitative reverse transcription-PCR assays indicated that deletion of hrpB reduced the expression of four type IV pili genes. The transcriptional start site of fimA (XAC3241) was determined using rapid amplification of 5′-cDNA Ends (5′RACE). Based on the results of fimA mRNA structure predictions, the fimA 5′ UTR may contain three different loops. HrpB may be involved in alterations to the structure of fimA mRNA that promote the stability of fimA RNA.ConclusionsOur data show that hrpB is involved in adherence of Xanthomonas citri subsp. citri to different surfaces. In addition, to the best of our knowledge, this is the first time that a DEAH RNA helicase has been implicated in the regulation of type IV pili in Xanthomonas.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0655-1) contains supplementary material, which is available to authorized users.

The unphosphorylated form of the PilR two-component system regulates pilA gene expression in Geobacter sulfurreducens

In Geobacter sulfurreducens, metal reduction and generation of bioelectricity require the participation of several elements, and among them, the type IV pili has an essential role. The pilus is composed of multiple PilA monomers. Expression of pilA gene depends mainly on the σ54 factor and the response regulator protein PilR. In this work, we characterized the role of the PilS-PilR two-component system in the regulation of the pilA gene expression. Experimental evidence indicates that PilS is autophosphorylated at the His-334 residue, which in turn is transferred to the conserved Asp-53 in PilR. Contrary to other PilS-PilR systems, substitution D53N in PilR resulted in higher activation of the pilA gene. By using a pilA::luxCDABE fusion with different promoter fragments and in vitro DNA-binding assays, we demonstrated the existence of multiple functional PilR binding sites. A regulatory model in which the non-phosphorylated PilR protein directs activation of pilA expression by binding to two sites in the promoter region of this gene is presented.

The pilT gene contributes to type III ExoS effector injection into epithelial cells in Pseudomonas aeruginosa.

A type IV pilus filament, mainly composed of PilA, is retracted by the driving power generated by PilT and PilU ATPases. pilA is required for injection of type III ExoS effectors into epithelial cells thereby facilitating Pseudomonas aeruginosa penetration through the epithelial barrier by impairing the defense function of tight junctions. Here, we examined whether the pilT and pilU of the P.aeruginosa PAO1 strain are required for ExoS injection into epithelial cells. We measured the quantity of ExoS injected into epithelial cells, and found that within such cells its quantity decreased by 80% (ΔpilA strain), 75% (ΔpilT strain), and 30% (ΔpilU strain) compared with the wild-type strain. pilT deficiency decreased the disruption of human epithelial colorectal adenocarcinoma (Caco-2) cell monolayers to the same extent as that of pilA and exoS deficiency, whereas pilU deficiency decreased disruption of the monolayers less than deficiency of the other genes. pilT and pilU deficiency decreased bacterial penetration of the Caco-2cell monolayers to the same level as pilA and exoS deficiency. Our data showed that the pilU gene expression level was reduced in the PAO1 strain after adhesion to Caco-2cell surfaces, but the expression levels of the pilA and pilT genes did not change. We conclude that P.aeruginosa injects ExoS into cells through the function of type IV pilus retraction, and that pilT makes a greater contribution to this process than pilU.

Genomic and Functional Characterization of the Unusual pLOCK 0919 Plasmid Harboring the spaCBA Pili Cluster in Lactobacillus casei LOCK 0919.

Here, we report the extensive bioinformatic and functional analyses of the unusual pLOCK 0919, a plasmid originating from the probiotic Lactobacillus casei LOCK 0919 strain. This plasmid is atypical because it harbors the spaCBA-srtC gene cluster encoding SpaCBA pili. We show that all other spaCBA-srtC sequences of the Lactobacillus genus that have been previously described and deposited in GenBank are present in the chromosomal DNA. Another important observation for pLOCK 0919 is that the spaCBA-srtC gene cluster and its surrounding genes are highly similar to the respective DNA region that is present in the most well-known and active SpaCBA pili producer, the probiotic Lactobacillus rhamnosus GG strain. Our results demonstrate that the spaCBA-srtC clusters of pLOCK 0919 and L. rhamnosus GG are genealogically similar, located in DNA regions that are rich in transposase genes and are poorly conserved among the publicly available sequences of Lactobacillus sp. In contrast to chromosomally localized pilus gene clusters from L. casei and Lactobacillus paracasei, the plasmidic spaC of L. casei LOCK 0919 is expressed and undergoes a slight glucose-induced repression. Moreover, results of series of in vitro tests demonstrate that L. casei LOCK 0919 has an adhesion potential, which is largely determined by the presence of the pLOCK 0919 plasmid. In particular, the plasmid occurrence positively influenced the hydrophobicity and aggregation abilities of L. casei LOCK 0919. Moreover, in vivo studies indicate that among the three Lactobacillus strains used to colonize the gastrointestinal tract of germ-free mice, already after 2 days of colonization, L. casei LOCK 0919 became the dominant strain and persisted there for at least 48 days.

Evidence that a modified type IV pilus-like system powers gliding motility and polysaccharide secretion in filamentous cyanobacteria.

In filamentous cyanobacteria, the mechanism of gliding motility is undefined but posited to be driven by a polysaccharide secretion system known as the junctional pore complex (JPC). Recent evidence implies that the JPC is a modified type IV pilus-like structure encoded for in part by genes in the hps locus. To test this hypothesis, we conducted genetic, cytological and comparative genomics studies on hps and pil genes in Nostoc punctiforme, a species in which motility is restricted to transiently differentiated filaments called hormogonia. Inactivation of most hps and pil genes abolished motility and abolished or drastically reduced secretion of hormogonium polysaccharide, and the subcellular localization of several Pil proteins in motile hormogonia corresponds to the site of the junctional pore complex. The non-motile ΔhpsE-G strain, which lacks three glycosyltransferases that synthesize hormogonium polysaccharide, could be complemented to motility by the addition of medium conditioned by wild-type hormogonia. Based on this result, we speculate that secretion of hormogonium polysaccharide facilitates but does not provide the motive force for gliding. Both the Hps and Pil homologs characterized in this study are almost universally conserved among filamentous cyanobacteria, with the Hps homologs rarely found in unicellular strains. These results support the theory that Hps and Pil proteins compose the JPC, a type IV pilus-like nanomotor that drives motility and polysaccharide secretion in filamentous cyanobacteria.

H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae.

Initially, pilE transcription in Neisseria gonorrhoeae appeared to be complicated, yet it was eventually simplified into a model where integration host factor activates a single -35/ -10 promoter. However, with the advent of high-throughput RNA sequencing, numerous small pil-specific RNAs (sense as well as antisense) have been identified at the pilE locus as well as at various pilS loci. Using a combination of in vitro transcription, site-directed mutagenesis, Northern analysis and quantitative reverse transcriptase PCR (qRT-PCR) analysis, we have identified three additional non-canonical promoter elements within the pilE gene; two are located within the midgene region (one sense and one antisense), with the third, an antisense promoter, located immediately downstream of the pilE ORF. Using strand-specific qRT-PCR analysis, an inverse correlation exists between the level of antisense expression and the amount of sense message. By their nature, promoter sequences tend to be AT-rich. In Escherichia coli, the small DNA-binding protein H-NS binds to AT-rich sequences and inhibits intragenic transcription. In N. gonorrhoeae hns mutants, pilE antisense transcription was increased twofold, with a concomitant decrease in sense transcript levels. However, most noticeably in these mutants, the absence of H-NS protein caused pilE/pilS recombination to increase dramatically when compared with WT values. Consequently, H-NS protein suppresses pilE intragenic transcription as well as antigenic variation through the pilE/pilS recombination system.