Pilus Type Research Articles

Transport and Polymerization of Porphyromonas gingivalis Type V Pili.

Adhesive pili (or fimbriae) in bacteria are classified into five types, among which type V pili have been most recently described. Type V pili differ from other pili types with respect to transport mechanism, structure, and pilin synthesis. Genes of type V pili are restricted to the phylum Bacteroidetes. Protein subunits that compose type V pili are transported to the cell surface as lipoprotein precursors and then polymerized into a pilus through a strand-exchange mechanism, which is demonstrated by several experiments, including palmitic acid labeling and Cys-Cys cross-linking analysis. Here, we describe the use of these methods to analyze type V pili.

The impact of PCV7/13 on the distribution of carried pneumococcal serotypes and on pilus prevalence; 14 years of repeated cross-sectional surveillance

BackgroundS. pneumoniae carriage by children is a major source of pneumococcal transmission, and the initial step prior to infection. Pilus type 1, reported in ~30% of pneumococcal strains in the pre-vaccine era, contributes to pneumococcal colonization and virulence. In this study, we report the impact of the pneumococcal conjugate vaccine (PCV), PCV7/PCV13 sequential implementation on serotype distribution, and on the prevalence of piliated strains among carried pneumococci during the pre- and post-vaccine eras. MethodsDuring 2002-2016, 12 repeated cross-sectional surveillances of nasopharyngeal S. pneumoniae carriage were conducted among 8,473 children <5.5 years old visiting primary care physicians in Central Israel. Seven biannual surveillances in the pre-PCV period, 2 surveillances after PCV7 was licensed but before implementation in the National Immunization Plan, and 3 additional surveillances in the post-PCV period. S. pneumoniae serotype distribution and prevalence of piliated strains were assessed. ResultsCarriage of S. pneumoniae was relatively stable (45.4%). The prevalence of serotypes included in PCV13 was 65.7%, in the pre-vaccine period and the pilus was present in 26.4% of isolates. The distribution of serotypes and the pilus prevalence in the pre-PCV period was relatively stable except for a decrease in prevalence of piliated 19F, observed following the first study year. Following PCV7/PCV13 implementation, vaccine type 13 (VT13) strains were nearly eliminated to 3.3% by 2016. Piliated strains, which were primarily of VT13 serotypes, initially followed a similar trend and were nearly eliminated by 2014 (1.7%). Yet, two years later, pilus prevalence re-emerged among non-VT strains to 12.8% of all pneumococci. ConclusionsFollowing PCV implementation, a dramatic and rapid decrease in VT strains prevalence was observed with a concomitant increase in non-VT strains. Piliated strains were nearly eliminated, yet re-emerged 7 years following PCV7/PCV13 implementation in various non-VT strains. This suggests that the pilus confers an advantage in colonization.

Expression of Ralstonia solanacearum type III secretion system is dependent on a novel type 4 pili (T4P) assembly protein (TapV) but is T4P independent.

Type IV pili (T4P) are virulence factors in various pathogenic bacteria of animals and plants that play important roles in twitching motility, swimming motility, biofilm formation, and adhesion to host cells. Here, we genetically characterized functional roles of a putative T4P assembly protein TapV (Rsc1986 in reference strain GMI1000) and its homologue Rsp0189, which shares 58% amino acid identity with TapV, in Ralstonia solanacearum. Deletion of tapV, but not rsp0189, resulted in significantly impaired twitching motility, swimming motility, and adhesion to tomato roots, which are consistent as phenotypes of the pilA mutant (a known R. solanacearum T4P‐deficient mutant). However, unlike the pilA mutant, the tapV mutant produced more biofilm than the wild‐type strain. Our gene expression studies revealed that TapV, but not Rsp0189, is important for expression of a type III secretion system (T3SS, a pathogenicity determinant of R. solanacearum) both in vitro and in planta, but it is T4P independent. We further revealed that TapV affected the T3SS expression via the PhcA–TapV–PrhG–HrpB pathway, consistent with previous reports that PhcA positively regulates expression of pilA and prhG. Moreover, deletion of tapV, but not rsp0189, significantly impaired the ability to migrate into and colonize xylem vessels of host plants, but there was no alteration in intercellular proliferation of R. solanacearum in tobacco leaves, which is similar to the pilA mutant. The tapV mutant showed significantly impaired virulence in host plants. This is the first report on the impact of T4P components on the T3SS, providing novel insights into our understanding of various biological functions of T4P and the complex regulatory pathway of T3SS in R. solanacearum.

The impact of the ancillary pilus-1 protein RrgA of Streptococcus pneumoniae on colonization and disease.

The Gram-positive bacterium Streptococcus pneumoniae, the pneumococcus, is an important commensal resident of the human nasopharynx. Carriage is usually asymptomatic, however, S. pneumoniae can become invasive and spread from the upper respiratory tract to the lungs causing pneumonia, and to other organs to cause severe diseases such as bacteremia and meningitis. Several pneumococcal proteins important for its disease-causing capability have been described and many are expressed on the bacterial surface. The surface located pneumococcal type-1 pilus has been associated with virulence and the inflammatory response, and it is present in 20%-30% of clinical isolates. Its tip protein RrgA has been shown to be a major adhesin to human cells and to promote invasion through the blood-brain barrier. In this review we discuss recent findings of the impact of RrgA on bacterial colonization of the upper respiratory tract and on pneumococcal virulence, and use epidemiological data and genome-mining to suggest trade-off mechanisms potentially explaining the rather low prevalence of pilus-1 expressing pneumococci in humans.

Delineation of a Subgroup of the Genus Paraburkholderia, Including P.terrae DSM 17804T, P.hospita DSM 17164T, and Four Soil-Isolated Fungiphiles, Reveals Remarkable Genomic and Ecological Features-Proposal for the Definition of a P.hospita Species Cluster.

The fungal-interactive (fungiphilic) strains BS001, BS007, BS110, and BS437 have previously been preliminarily assigned to the species Paraburkholderia terrae. However, in the (novel) genus Paraburkholderia, an as-yet unresolved subgroup exists, that clusters around Paraburkholderia hospita (containing the species P. terrae, P. hospita, and Paraburkholderia caribensis). To shed light on the precise relationships across the respective type strains and the novel fungiphiles, we here compare their genomic and ecophysiological features. To reach this goal, the genomes of the three type strains, with sizes ranging from 9.0 to 11.5 Mb, were de novo sequenced and the high-quality genomes analyzed. Using whole-genome, ribosomal RNA and marker-gene-concatenate analyses, close relationships between P. hospita DSM 17164T and P. terrae DSM 17804T, versus more remote relationships to P. caribensis DSM 13236T, were found. All four fungiphilic strains clustered closely to the two-species cluster. Analyses of average nucleotide identities (ANIm) and tetranucleotide frequencies (TETRA) confirmed the close relationships between P. hospita DSM 17164T and P. terrae DSM 17804T (ANIm = 95.42; TETRA = 0.99784), as compared with the similarities of each one of these strains to P. caribensis DSM 13236T. A species cluster was thus proposed. Furthermore, high similarities of the fungiphilic strains BS001, BS007, BS110, and BS437 with this cluster were found, indicating that these strains also make part of it, being closely linked to P. hospita DSM 17164T (ANIm = 99%; TETRA = 0.99). We propose to coin this cluster the P. hospita species cluster (containing P. hospita DSM 17164T, P. terrae DSM 17804T, and strains BS001, BS007, BS110, and BS437), being clearly divergent from the closely related species P. caribensis (type strain DSM 13236T). Moreover, given their close relatedness to P. hospita DSM 17164T within the cluster, we propose to rename the four fungiphilic strains as members of P. hospita. Analysis of migratory behavior along with fungal growth through soil revealed both P. terrae DSM 17804T and P. hospita DSM 17164T (next to the four fungiphilic strains) to be migration-proficient, whereas P. caribensis DSM 13236T was a relatively poor migrator. Examination of predicted functions across the genomes of the seven investigated strains, next to several selected additional ones, revealed the common presence of features in the P. hospita cluster strains that are potentially important in interactions with soil fungi. Thus, genes encoding specific metabolic functions, biofilm formation (pelABCDEFG, pgaABCD, alginate-related genes), motility/chemotaxis, type-4 pili, and diverse secretion systems were found.

Filament genes and biofilm formation in Streptococcus agalactiae

Streptococcus agalactiae is the prominent cause of bacterial sepsis and meningitis in neonates and is also the contributing agent of a number of serious infections in immunocompromised adults. S. agalactiae run into multiple niches through an infection, suggesting that regulatory mechanisms control the expression of specific virulence factors in this bacterium. Like other Gram-positive bacteria, Group B streptococcus (GBS) can create three-dimensional structures, such as biofilms that can increase their ability to colonize and maintain in the host. Biofilm formation has been investigated by GBS under laboratory conditions and is clearly controlled under environmental conditions. Significantly, antibodies that act against the proteins of the pili can prevent the creation of biofilms. The consequences of biofilm formation in the field of asymptomatic colonization and its dissemination remain invasive and its details are examined. The complex pathway leading to biofilm development in different species of microorganisms involves the contribution of both environmental conditions and genetic factors. Numerous genes or factors have been identified as being essential or required for biofilm formation. Such genes include those that regulate surface-exposed proteins, appendages, such as pili or fimbriae, and extracellular polymeric substance (EPS) matrix materials. Pilus seems to play a key role in adhesion and attachment to host cells both in Gram-negative and Gram-positive pathogens. Their involvement in the transition from planktonic growth to a surface-attached multicellular community has also been demonstrated in many studies. Three different types of pili have been characterized in GBS as potential virulence factors. For example, type 2a pili are involved in biofilm formation.

Molecular Characterization of Hospital- and Community-Acquired Streptococcus agalactiae Isolates among Nonpregnant Adults in Isfahan, Iran.

Background:The increasing incidence of Group B Streptococcus (GBS) infection among nonpregnant adults has become of growing clinical and public health concern. The current study investigated the distribution of important virulence determinants and antibiotic susceptibility of GBS isolates causing community acquired (CA) and hospital acquired (HA) infections among nonpregnant adults.Materials and Methods:A total of 62 GBS, including 31 CA GBS and 31 HA GBS, were collected from a teaching hospital in Isfahan, Iran. Capsular polysaccharide genotypes (CPS), PI 1, PI 2a, PI 2b, and hypervirulent GBS adhesin (hvgA) virulence genes and antibiotic resistance profiling were determined.Results:There were 19 (30.6%) cases of underlying disease that diabetes mellitus (20.9%) was most common. The rate of multidrug resistant GBS strains was accounted for 29%. Distribution of macrolide resistant phenotypes was as follows: constitutive macrolides, lincosamides, and streptogramin B (MLSB) (15 isolates); inducible resistance to MLSB; and L phenotype (each 5 isolates) and M phenotype (1 isolate). V and Ia serotypes were the most predominant capsular type in HA GBS and CA GBS isolates, respectively. The most frequent pilus types were PI 1, PI 1+PI 2a, PI 1+PI 2b, and PI 2a. PI 1 and PI 1+PI 2a had significantly different distributions between CA and HA GBS isolates. Three CA GBS isolates (9.6%) were positive for hvgA gene that belonged to clonal complex 17/sequence type 17/CPS III/PI 1+PI 2b lineage.Conclusion:There was a significant difference in the distribution of PIs among CA GBS and HA GBS isolates in our region.

Characterization and Bio-Typing of Multidrug Resistance Plasmids From Uropathogenic Escherichia coli Isolated From Clinical Setting.

Urinary tract infection is primarily caused by Escherichia coli. Multidrug resistance and their rapid dissemination in this pathogenic microbe complicate therapeutic strategies and threaten public health. Conjugation systems responsible for interbacterial transmission of antibiotic resistance are plasmid-encoded and can be classified as the P, F, and I types. Specific pili types and pili associated proteins were related to the transfer among this gram-negative organism and were thought to depend on contacts created by these structures at the time of DNA transport. In this study, conjugation system types of the plasmids that harbor multidrug resistant genes (aac-1b-cr, oqxAB, qnrB, qnrS, blaTEM, blaOXA) amongst 19 E. coli uropathogenic isolates were characterized under ciprofloxacin/ceftazidime selection individually by pili and pili associated gene types. Investigations indicated incidence of single plasmid of multiple replicon type amongst the transconjugants. blaTEM, blaCTX–M, blaOXA, aac-1b-cr, oqxAB, qnrB, qnrS genes in varied combination were observed to be successfully co-transmitted against ceftazidme/ciprofloxacin selection. Seven primer pair sets were selected that encodes pili and pili associated genes (traF, trwJ, traE, trhE, traG, pilM, pilx4) by nucleotide database search tools using annotated plasmids of different incompatibility types to assign the conjugation system type of the transmissible resistant plasmids by PCR. traF was predominant irrespective of drug selection that indicated F-type conjugation system was responsible for transmission of resistant plasmids which results in the rapid dissemination of antibiotic resistance in the isolates screened. Therefore this is a first report of its kind that investigated pili and pili associated genes to bio-type multidrug resistant plasmids and their transmission in clinical settings amongst uropathogenic E. coli circulated in the eastern part of India.

Prevalence of PspA families and pilus islets among Streptococcus pneumoniae colonizing children before and after universal use of pneumococcal conjugate vaccines in Brazil

In 2010, the 10-valent (PCV10) and 13-valent (PCV13) pneumococcal conjugate vaccines were introduced in Brazil to immunize children, resulting in serotype replacement. We analyzed 253 carriage isolates recovered from children aged <6 years in Brazil, including 124 and 129 isolates from the pre-PCV10/13 (December 2009-July 2010) and post-PCV10/13 (September-December 2014) periods, respectively, to investigate the prevalence of PspA families and pilus islets, potential vaccine candidates. Serotypes and resistance profiles were previously characterized. We used PCR to type PspA families (Fam1-3) and pilus islets (PI-1 and PI-2). We identified the PspA family of 130 (51.4%) isolates. PspA families 1, 2, and 3 were identified in 12.2%, 38.7%, and 0.4% of the isolates, respectively. Eighteen (58.1%) Fam1 isolates were serogroup 6. Nine (81.8%) of 11 serotype 14 isolates were Fam2. Fam1 isolates resistant to penicillin (50%), erythromycin (43.7%), clindamycin (31.2%), and chloramphenicol (6.2%) were only found after PCV10/13 introduction. Resistance among Fam2 isolates was higher in the post-PCV10/13 period to erythromycin (1.8% vs. 18.6%), clindamycin (0 vs. 13.9%), and tetracycline (10.9% vs. 16.3%). PI-I was detected in 42 (16.6%) isolates. Fourteen (56%) of 25 serotype 15B/C and nine (81.8%) of 11 serotype 14 isolates had PI-1 (p < 0.01). Eight (3.2%) isolates had PI-2, and six (75%) were serogroup 19. Five (2%) serogroup 19 isolates had both PI-1 and PI-2. We found associations between serogroups/serotypes, PspA families, and pilus islets, but distribution of PspA families and pilus islets was similar in both periods. After universal vaccination, we observed higher antimicrobial resistance frequencies, regardless PspA or pilus types.

Genomic characterisation of perinatal Western Australian Streptococcus agalactiae isolates.

As a leading cause of neonatal sepsis, Streptococcus agalactiae, commonly known as Group B Streptococcus, is a major neonatal pathogen. Current global screening practices employ risk- or culture-based protocols for detection of these organisms. In Western Australia (WA), universal culture-based screening is provided, with subsequent intrapartum antibiotic prophylaxis for all S. agalactiae-positive women during labour. Widespread antibiotic exposure is not ideal and this is one of the factors driving development of vaccines against S. agalactiae. Vaccine candidates have focused on the capsule, surface proteins and pilus types, however, capsule serotypes are known to vary geographically. The aim of this study was to use genome sequencing to gain an understanding of the circulating genotypes in WA, and to assess variations in the associated gene pools. We sequenced 141 antenatal carriage (vaginal/rectal) isolates and 10 neonatal invasive disease isolates from WA. Based on the global PubMLST database, the 151 strains were characterised into 30 sequence types, with clustering of these mainly into clonal complexes 1, 12, 17, 19 and 23. Of the genes encoding eleven surface proteins that were analysed, the most prevalent were fbp, lmb and scpB which were present in ≥ 98% of isolates. A cluster of non-haemolytic isolates, one of which was a neonatal invasive disease isolate, appeared to lack the entire cyl locus. Admixture analysis of population structure revealed evidence of genetic transfer among the WA isolates across structural groups. When compared against the PubMLST S. agalactiae data, WA isolates showed high levels of strain diversity with minimal apparent clustering. This is the first whole genome sequence study of WA S. agalactiae isolates and also represents the first addition of Australian isolate data to PubMLST. This report provides insight into the distribution and diversity of vaccine targets of S. agalactiae within Western Australia, indicating that the most appropriate capsular vaccine for this population would be the proposed pentavalent (Cps Ia, Ib, II, III and V) preparation, whilst vaccines targeting surface proteins should ideally utilise Fbp, Lmb and/or ScpB.

Multistate population and whole genome sequence-based strain surveillance of invasive pneumococci recovered in the USA during 2017

ObjectivesWe aimed to provide population-based and whole-genome sequence (WGS) -based characterization of invasive pneumococcal disease isolates collected from multistate surveillance in the USA during 2017. MethodsWe obtained short-read WGS from 2881 isolates with associated bioinformatics pipeline strain feature predictions. For quality control, capsular serotypes and antimicrobial MICs were also obtained conventionally from 442 isolates. Annotated WGS were provided (inclusive of serotypes, MICs, multilocus sequence types, pilus type(s)) from 2723 isolates. For 158 isolates with suboptimal WGS, antimicrobial MICs were obtained conventionally. ResultsThere were 127 isolates from children <5 years of age and 2754 isolates from those ≥5 years old in 2017. One of 43 different serotypes was predicted for 2877 of the 2881 isolates. Serotypes in the 13-valent conjugate vaccine together with 6C (PCV13+6C) accounted for 816 (28.3%) isolates, with PCV13 serotype 3 being the most common serotype overall. Non-PCV13–6C– serotypes accounted for 2065 (71.7%) isolates, comprising 96 (75.6%) isolates from children < 5 years old and 1969 (61.4%) isolates from those aged ≥5 years. Of 36 different categories of recently emerged serotype-switch variants, three showed marked increases relative to 2015–2016 in that the number from 2017 surpassed the number from 2015–2016 combined. Two of these included antimicrobial-resistant serotype 11A and 35B serotype-switch variants of the ST156 clonal complex. ConclusionsPCV13+6C strains are still identified in 2017 but non-PCV13-type strains impose a considerable burden. This well-annotated year 2017 WGS/strain data set will prove useful for a broad variety of analyses and improved our understanding of invasive pneumococcal disease-causing strains in the post-PCV13 era.

Comparative genomics of Aeromonas veronii: Identification of a pathotype impacting aquaculture globally.

Aeromonas veronii is a gram-negative species abundant in aquatic environments that causes disease in humans as well as terrestrial and aquatic animals. In the current study, 41 publicly available A. veronii genomes were compared to investigate distribution of putative virulence genes, global dissemination of pathotypes, and potential mechanisms of virulence. The complete genome of A. veronii strain ML09-123 from an outbreak of motile aeromonas septicemia in farm-raised catfish in the southeastern United States was included. Dissemination of A. veronii strain types was discovered in dispersed geographical locations. Isolate ML09-123 is highly similar to Chinese isolate TH0426, suggesting the two strains have a common origin and may represent a pathotype impacting aquaculture in both countries. Virulence of strain ML09-123 in catfish in a dose-dependent manner was confirmed experimentally. Subsystem category disposition showed the majority of genomes exhibit similar distribution of genomic elements. The type I secretion system (T1SS), type II secretion system (T2SS), type 4 pilus (T4P), and flagellum core elements are conserved in all A. veronii genomes, whereas the type III secretion system (T3SS), type V secretion system (T5SS), type VI secretion system (T6SS), and tight adherence (TAD) system demonstrate variable dispersal. Distribution of mobile elements is dependent on host and geographic origin, suggesting this species has undergone considerable genetic exchange. The data presented here lends insight into the genomic variation of A. veronii and identifies a pathotype impacting aquaculture globally.

Motor Properties of PilT-Independent Type 4 Pilus Retraction in Gonococci.

Bacterial type 4 pili (T4P) belong to the strongest molecular machines. The gonococcal T4P retraction ATPase PilT supports forces exceeding 100 pN during T4P retraction. Here, we address the question of whether gonococcal T4P retract in the absence of PilT. We show that pilT deletion strains indeed retract their T4P, but the maximum force is reduced to 5 pN. Similarly, the speed of T4P retraction is lower by orders of magnitude compared to that of T4P retraction driven by PilT. Deleting the pilT paralogue pilT2 further reduces the speed of T4P retraction, yet T4P retraction is detectable in the absence of all three pilT paralogues. Furthermore, we show that depletion of proton motive force (PMF) slows but does not inhibit pilT-independent T4P retraction. We conclude that the retraction ATPase is not essential for gonococcal T4P retraction. However, the force generated in the absence of PilT is too low to support important functions of T4P, including twitching motility, fluidization of colonies, and induction of host cell response.IMPORTANCE Bacterial type 4 pili (T4P) have been termed the "Swiss Army knives" of bacteria because they perform numerous functions, including host cell interaction, twitching motility, colony formation, DNA uptake, protein secretion, and surface sensing. The pilus fiber continuously elongates or retracts, and these dynamics are functionally important. Curiously, only a subset of T4P systems employ T4P retraction ATPases to power T4P retraction. Here, we show that one of the strongest T4P machines, the gonococcal T4P, retracts without a retraction ATPase. Biophysical characterization reveals strongly reduced force and speed compared to retraction with ATPase. We propose that bacteria encode retraction ATPases when T4P have to generate high-force-supporting functions like twitching motility, triggering host cell response, or fluidizing colonies.

A Putative Type V Pilus Contributes to Bacteroides thetaiotaomicron Biofilm Formation Capacity.

Bacteroides thetaiotaomicron is a prominent anaerobic member of the healthy human gut microbiota. While the majority of functional studies on B. thetaiotaomicron addressed its impact on the immune system and the utilization of diet polysaccharides, B. thetaiotaomicron biofilm capacity and its contribution to intestinal colonization are still poorly characterized. We tested the natural adhesion of 34 B. thetaiotaomicron isolates and showed that although biofilm capacity is widespread among B. thetaiotaomicron strains, this phenotype is masked or repressed in the widely used reference strain VPI 5482. Using transposon mutagenesis followed by a biofilm positive-selection procedure, we identified VPI 5482 mutants with increased biofilm capacity corresponding to an alteration in the C-terminal region of BT3147, encoded by the BT3148-BT3147 locus, which displays homology with Mfa-like type V pili found in many Bacteroidetes We show that BT3147 is exposed on the B. thetaiotaomicron surface and that BT3147-dependent adhesion also requires BT3148, suggesting that BT3148 and BT3147 correspond to the anchor and stalk subunits of a new type V pilus involved in B. thetaiotaomicron adhesion. This study therefore introduces B. thetaiotaomicron as a model to study proteinaceous adhesins and biofilm-related phenotypes in this important intestinal symbiont.IMPORTANCE Although the gut anaerobe Bacteroides thetaiotaomicron is a prominent member of the healthy human gut microbiota, little is known about its capacity to adhere to surfaces and form biofilms. Here, we identify that alteration of a surface-exposed protein corresponding to a type of pili found in many Bacteroidetes increases B. thetaiotaomicron biofilm formation. This study lays the ground for establishing this bacterium as a model organism for in vitro and in vivo studies of biofilm-related phenotypes in gut anaerobes.

Dynamics of a type 2 secretion system pseudopilus unraveled by complementary approaches

Secretion pili, bacterial fibers responsible for transporting proteins to the extracellular milieu in some secretion systems, are very strong structures but at the same time highly flexible. Their flexibility and helical symmetry make structure determination at atomic resolution a challenging task. We have previously used an integrative structural biology approach including liquid-state NMR, cryo-electron microscopy (cryo-EM), and modeling to determine the pseudo-atomic resolution structure of the type 2 secretion system pseudopilus in a mutant form, where we employed NMR to determine the high resolution structure of the pilin (the monomer building block of the pilus). In this work, we determine the pseudo-atomic structure of the wild type pilus, and compare the dynamics of wild type and mutant pili by normal mode analysis. We present a detailed NMR analysis of the dynamics of the pilin in isolation, and compare dynamics and solvent accessibility of isolated and assembled pilins by Hydrogen/Deuterium eXchange Mass Spectrometry (HDX-MS). These complementary approaches provide a comprehensive view of internal and overall dynamics of pili, crucial for their function.

Small RNA AvrA Regulates IscR to Increase the Stress Tolerances in SmpB Deficiency of Aeromonas veronii.

The superbacteria Aeromonas veronii displays not only a strong pathogenicity but also the resistance to nine kinds of antibiotics, resulting in the economic losses and health hazards. Small Protein B (SmpB) plays an important role in protein quality control, virulence, and stress reactions. Transcriptomic data revealed that expressions of the type IV pilus assembly and type VI secretion system (T6SS) proteins were downregulated in SmpB deficiency, indicating that the virulence of A. veronii might be attenuated. Although SmpB deletion decreased colonization in the mouse spleen and liver, LD50 of the smpB mutant was not altered as expected, compared with the wild type. Further, the transcriptomic and quantitative RT-PCR analyses showed that the combination of the downregulated AvrA and the upregulated iron-sulfur protein activator IscR, mediated the oxidative tolerance in smpB deletion. Next a reporter plasmid was constructed in which the promoter of iscR was applied to control the expression of the enhanced green fluorescent protein (eGFP) gene. When the reporter plasmid was co-expressed with the AvrA expression into E. coli, the relative fluorescence intensity was decreased significantly, suggesting that AvrA bound to iscR mRNA by base pairing, which in turn relieved the inhibition of iscR and intensified the downstream iron-sulfur proteins. Collectively, the smpB mutant exhibited an attenuated virulence in mice and enhanced tolerances to oxidative stress. This study demonstrates the complexity of gene regulation networks mediated by sRNA in systems biology, and also reflects the strong adaptability of superbacteria A. veronii in the process of evolution.

Characterization of the Citrobacter rodentium Cpx regulon and its role in host infection.

Envelope-localized proteins, such as adhesins and secretion systems, play critical roles in host infection by Gram-negative pathogens. As such, their folding is monitored by envelope stress response systems. Previous studies demonstrated that the Cpx envelope stress response is required for virulence of Citrobacter rodentium, a murine pathogen used to model infections by the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli; however, the mechanisms by which the Cpx response promotes host infection were previously unknown. Here, we characterized the C. rodentium Cpx regulon in order to identify genes required for host infection. Using transcriptomic and proteomic approaches, we found that the Cpx response upregulates envelope-localized protein folding and degrading factors but downregulates pilus genes and type III secretion effectors. Mouse infections with C. rodentium strains lacking individual Cpx-regulated genes showed that the chaperone/protease DegP and the disulfide bond oxidoreductase DsbA were essential for infection, but Cpx regulation of these genes did not fully account for attenuation of C. rodentium ΔcpxRA. Both deletion of dsbA and treatment with the reducing agent dithiothreitol activated the C. rodentium Cpx response, suggesting that it may sense disruption of disulfide bonding. Our results highlight the importance of envelope protein folding in host infection by Gram-negative pathogens.

Development of a multiplex PCR assay based on the pilA gene sequences to detect different types of Acidovorax citrulli

Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax citrulli, is a major threat to commercial watermelon and melon production worldwide. At present, there are at least two genetically distinct sub-populations (group I and II) of A. citrulli that differ in host preference among cucurbit species and copper sensitivity. In this study, we analyzed the pilA gene sequences of 103 A. citrulli strains from China and other countries. Based on these data, we classified all tested A. citrulli strains into three types. The pilA-based type 1 strains in this study coincided with the previously established group I strains; while the type 2 strains coincided with group II strains. Ten strains that did not cluster with group I or II strains were classified into a new type, designated type 3. Based on differences in pilA sequences, we designed a multiplex PCR assay to distinguish the three A. citrulli pilus types. This multiplex PCR assay has proven to be viable for strain typing of 139 A. citrulli strains and for the detection of this pathogen in artificially inoculated seeds and leaves and naturally infected leaves and fruits. This assay proved to be rapid, accurate, reliable and applicable for early distinction of A. citrulli types associated with BFB epidemics. It may also inform the judicious and environmentally sound use of bactericides, especially copper-based compounds.

Type IV Pilin Post-Translational Modifications Modulate Material Properties of Bacterial Colonies

Bacterial type 4 pili (T4P) are extracellular polymers that initiate the formation of microcolonies and biofilms. T4P continuously elongate and retract. These pilus dynamics crucially affect the local order, shape, and fluidity of microcolonies. The major pilin subunit of the T4P bears multiple post-translational modifications. By interfering with different steps of the pilin glycosylation and phosphoform modification pathways, we investigated the effect of pilin post-translational modification on the shape and dynamics of microcolonies formed by Neisseria gonorrhoeae. Deleting the phosphotransferase responsible for phosphoethanolamine modification at residue serine 68 inhibits shape relaxations of microcolonies after perturbation and causes bacteria carrying the phosphoform modification to segregate to the surface of mixed colonies. We relate these mesoscopic phenotypes to increased attractive forces generated by T4P between cells. Moreover, by deleting genes responsible for the pilin glycan structure, we show that the number of saccharides attached at residue serine 63 affects the ratio between surface tension and viscosity and cause sorting between bacteria carrying different pilin glycoforms. We conclude that different pilin post-translational modifications moderately affect the attractive forces between bacteria but have severe effects on the material properties of microcolonies.

Novel type of pilus associated with a Shiga-toxigenic E. coli hybrid pathovar conveys aggregative adherence and bacterial virulence

A large German outbreak in 2011 was caused by a locus of enterocyte effacement (LEE)-negative enterohemorrhagic E. coli (EHEC) strain of the serotype O104:H4. This strain harbors markers that are characteristic of both EHEC and enteroaggregative E. coli (EAEC), including aggregative adhesion fimbriae (AAF) genes. Such rare EHEC/EAEC hybrids are highly pathogenic due to their possession of a combination of genes promoting severe toxicity and aggregative adhesion. We previously identified novel EHEC/EAEC hybrids and observed that one strain exhibited aggregative adherence but had no AAF genes. In this study, a genome sequence analysis showed that this strain belongs to the genoserotype O23:H8, MLST ST26, and harbors a 5.2 Mb chromosome and three plasmids. One plasmid carries some EAEC marker genes, such as aatA and genes with limited protein homology (11–61%) to those encoding the bundle-forming pilus (BFP) of enteropathogenic E. coli. Due to significant protein homology distance to known pili, we designated these as aggregate-forming pili (AFP)-encoding genes and the respective plasmid as pAFP. The afp operon was arranged similarly to the operon of BFP genes but contained an additional gene, afpA2, which is homologous to afpA. The deletion of the afp operon, afpA, or a nearby gene (afpR) encoding an AraC-like regulator, but not afpA2, led to a loss of pilin production, piliation, bacterial autoaggregation, and importantly, a >80% reduction in adhesion and cytotoxicity toward epithelial cells. Gene sets similar to the afp operon were identified in a variety of aatA-positive but AAF-negative intestinal pathogenic E. coli. In summary, we characterized widely distributed and novel fimbriae that are essential for aggregative adherence and cytotoxicity in a LEE-negative Shiga-toxigenic hybrid.