The role of integrating conjugative elements in <Emphasis Type="Italic">Helicobacter pylori</Emphasis>: a review

The genome of Helicobacter pylori contains many putative genes, including a genetic region known as the Integrating Conjugative Elements of H. pylori type four secretion system (ICEHptfs). This genetic regions were originally termed as “plasticity zones/regions” due to the great genetic diversity between the original two H. pylori whole genome sequences. Upon analysis of additional genome sequences, the regions were reported to be extremely common within the genome of H. pylori. Moreover, these regions were also considered conserved rather than genetically plastic and were believed to act as mobile genetic elements transferred via conjugation. Although ICEHptfs(s) are highly conserved, these regions display great allele diversity, especially on ICEHptfs4, with three different subtypes: ICEHptfs4a, 4b, and 4c. ICEHptfs were also reported to contain a novel type 4 secretion system (T4SS) with both epidemiological and in vitro infection model studies highlighting that this novel T4SS functions primarily as a virulence factor. However, there is currently no information regarding the structure, the genes responsible for forming the T4SS, and the interaction between this T4SS and other virulence genes. Unlike the cag pathogenicity island (PAI), which contains CagA, a gene found to be essential for H. pylori virulence, these novel T4SSs have not yet been reported to contain genes that contribute significant effects to the entire system. This notion prompted the hypothesis that these novel T4SSs may have different mechanisms involving cag PAI.     

Mobile Antibiotic Resistance Encoding Elements Promote Their Own Diversity

Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter–ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage λ Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.     

Novel type IV secretion system involved in propagation of genomic islands

Type IV secretion systems (T4SSs) mediate horizontal gene transfer, thus contributing to genome plasticity, evolution of infectious pathogens, and dissemination of antibiotic resistance and other virulence traits. A gene cluster of the Haemophilus influenzae genomic island ICEHin1056 has been identified as a T4SS involved in the propagation of genomic islands. This T4SS is novel and evolutionarily distant from the previously described systems. Mutation analysis showed that inactivation of key genes of this system resulted in a loss of phenotypic traits provided by a T4SS. Seven of 10 mutants with a mutation in this T4SS did not express the type IV secretion pilus. Correspondingly, disruption of the genes resulted in up to 100,000-fold reductions in conjugation frequencies compared to those of the parent strain. Moreover, the expression of this T4SS was found to be positively regulated by one of its components, the tfc24 gene. We concluded that this gene cluster represents a novel family of T4SSs involved in propagation of genomic islands.     

Campylobacter fetus Subspecies Contain Conserved Type IV Secretion Systems on Multiple Genomic Islands and Plasmids

The features contributing to differences in pathogenicity of the Campylobacter fetus subspecies are unknown. Putative factors involved in pathogenesis are located in genomic islands that encode a type IV secretion system (T4SS) and fic domain (filamentation induced by cyclic AMP) proteins, which may disrupt host cell processes. In the genomes of 27 C. fetus strains, three phylogenetically-different T4SS-encoding regions (T4SSs) were identified: one was located in both the chromosome and in extra-chromosomal plasmids; one was located exclusively in the chromosome; and one exclusively in extra-chromosomal plasmids. We observed that C. fetus strains can contain multiple T4SSs and that homologous T4SSs can be present both in chromosomal genomic islands (GI) and on plasmids in the C. fetus strains. The GIs of the chromosomally located T4SS differed mainly by the presence of fic genes, insertion sequence elements and phage-related or hypothetical proteins. Comparative analysis showed that T4SS sequences, inserted in the same locations, were conserved in the studied C. fetus genomes. Using phylogenetic analysis of the T4SSs, it was shown that C. fetus may have acquired the T4SS regions from other Campylobacter species by horizontal gene transfer. The identified T4SSs and fic genes were found in Cff and Cfv strains, although the presence of T4SSs and fic genes were significantly associated with Cfv strains. The T4SSs and fic genes could not be associated with S-layer serotypes or geographical origin of the strains.     

Type III Secretion System and Virulence Markers Highlight Similarities and Differences between Human- and Plant-Associated Pseudomonads Related to Pseudomonas fluorescens and P. putida

Pseudomonas fluorescens is commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the related Pseudomonas aeruginosa nosocomial species and the occurrence of this secretion system in plant-associated P. fluorescens raise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging to P. fluorescens were compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41°C) isolates from patients suffering from blood infections; these isolates were finally found to not belong to P. fluorescens but were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs from P. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged to P. fluorescens and harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associated P. fluorescens.     

Small-molecule type III secretion system inhibitors block assembly of the Shigella type III secreton

Type III secretion systems (T3SSs) are essential virulence devices for many gram-negative bacteria that are pathogenic for plants, animals, and humans. They serve to translocate virulence effector proteins directly into eukaryotic host cells. T3SSs are composed of a large cytoplasmic bulb and a transmembrane region into which a needle is embedded, protruding above the bacterial surface. The emerging antibiotic resistance of bacterial pathogens urges the development of novel strategies to fight bacterial infections. Therapeutics that rather than kill bacteria only attenuate their virulence may reduce the frequency or progress of resistance emergence. Recently, a group of salicylidene acylhydrazides were identified as inhibitors of T3SSs in Yersinia, Chlamydia, and Salmonella species. Here we show that these are also effective on the T3SS of Shigella flexneri, where they block all related forms of protein secretion so far known, as well as the epithelial cell invasion and induction of macrophage apoptosis usually demonstrated by this bacterium. Furthermore, we show the first evidence for the detrimental effect of these compounds on T3SS needle assembly, as demonstrated by increased numbers of T3S apparatuses without needles or with shorter needles. Therefore, the compounds generate a phenocopy of T3SS export apparatus mutants but with incomplete penetrance. We discuss why this would be sufficient to almost completely block the later secretion of effector proteins and how this begins to narrow the search for the molecular target of these compounds.     

Type VI secretion systems of plant-pathogenic Burkholderia glumae BGR1 play a functionally distinct role in interspecies interactions and virulence

In the environment, bacteria show close association, such as interspecies interaction, with other bacteria as well as host organisms. The type VI secretion system (T6SS) in gram-negative bacteria is involved in bacterial competition or virulence. The plant pathogen Burkholderia glumae BGR1, causing bacterial panicle blight in rice, has four T6SS gene clusters. The presence of at least one T6SS gene cluster in an organism indicates its distinct role, like in the bacterial and eukaryotic cell targeting system. In this study, deletion mutants targeting four tssD genes, which encode the main component of T6SS needle formation, were constructed to functionally dissect the four T6SSs in B. glumae BGR1. We found that both T6SS group_4 and group_5, belonging to the eukaryotic targeting system, act independently as bacterial virulence factors toward host plants. In contrast, T6SS group_1 is involved in bacterial competition by exerting antibacterial effects. The ΔtssD1 mutant lost the antibacterial effect of T6SS group_1. The ΔtssD1 mutant showed similar virulence as the wild-type BGR1 in rice because the ΔtssD1 mutant, like the wild-type BGR1, still has key virulence factors such as toxin production towards rice. However, metagenomic analysis showed different bacterial communities in rice infected with the ΔtssD1 mutant compared to wild-type BGR1. In particular, the T6SS group_1 controls endophytic plant-associated bacteria such as Luteibacter and Dyella in rice plants and may have an advantage in competing with endophytic plant-associated bacteria for settlement inside rice plants in the environment. Thus, B. glumae BGR1 causes disease using T6SSs with functionally distinct roles.     

Endofungal bacterium controls its host by an hrp type III secretion system

Burkholderia rhizoxinica and Rhizopus microsporus form a unique symbiosis in which intracellular bacteria produce the virulence factor of the phytopathogenic fungus. Notably, the host strictly requires endobacteria to sporulate. In this study, we show that the endofungal bacteria possess a type III secretion system (T3SS), which has a crucial role in the maintenance of the alliance. Mutants defective in type III secretion show reduced intracellular survival and fail to elicit sporulation of the host. Furthermore, genes coding for T3SS components are upregulated during cocultivation of the bacterial symbiont with their host. This is the first report on a T3SS involved in bacterial–fungal symbiosis. Phylogenetic analysis revealed that the T3SS represents a prototype of a clade of yet uncharacterized T3SSs within the hrp superfamily of T3SSs from plant pathogenic microorganisms. In a control experiment, we demonstrate that under laboratory conditions, rhizoxin production was not required for establishment of the symbiotic interaction.     

Mechanism and structure of the bacterial type IV secretion systems

The bacterial type IV secretion systems (T4SSs) translocate DNA and protein substrates to bacterial or eukaryotic target cells generally by a mechanism dependent on direct cell-to-cell contact. The T4SSs encompass two large subfamilies, the conjugation systems and the effector translocators. The conjugation systems mediate interbacterial DNA transfer and are responsible for the rapid dissemination of antibiotic resistance genes and virulence determinants in clinical settings. The effector translocators are used by many Gram-negative bacterial pathogens for delivery of potentially hundreds of virulence proteins to eukaryotic cells for modulation of different physiological processes during infection. Recently, there has been considerable progress in defining the structures of T4SS machine subunits and large machine subassemblies. Additionally, the nature of substrate translocation sequences and the contributions of accessory proteins to substrate docking with the translocation channel have been elucidated. A DNA translocation route through the Agrobacterium tumefaciens VirB/VirD4 system was defined, and both intracellular (DNA ligand, ATP energy) and extracellular (phage binding) signals were shown to activate type IV-dependent translocation. Finally, phylogenetic studies have shed light on the evolution and distribution of T4SSs, and complementary structure-function studies of diverse systems have identified adaptations tailored for novel functions in pathogenic settings. This review summarizes the recent progress in our understanding of the architecture and mechanism of action of these fascinating machines, with emphasis on the ‘archetypal’ A. tumefaciens VirB/VirD4 T4SS and related conjugation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Uncovering the prevalence and diversity of integrating conjugative elements in actinobacteria

Horizontal gene transfer greatly facilitates rapid genetic adaptation of bacteria to shifts in environmental conditions and colonization of new niches by allowing one-step acquisition of novel functions. Conjugation is a major mechanism of horizontal gene transfer mediated by conjugative plasmids and integrating conjugative elements (ICEs). While in most bacterial conjugative systems DNA translocation requires the assembly of a complex type IV secretion system (T4SS), in Actinobacteria a single DNA FtsK/SpoIIIE-like translocation protein is required. To date, the role and diversity of ICEs in Actinobacteria have received little attention. Putative ICEs were searched for in 275 genomes of Actinobacteria using HMM-profiles of proteins involved in ICE maintenance and transfer. These exhaustive analyses revealed 144 putative FtsK/SpoIIIE-type ICEs and 17 putative T4SS-type ICEs. Grouping of the ICEs based on the phylogenetic analyses of maintenance and transfer proteins revealed extensive exchanges between different sub-families of ICEs. 17 ICEs were found in Actinobacteria from the genus Frankia, globally important nitrogen-fixing microorganisms that establish root nodule symbioses with actinorhizal plants. Structural analysis of ICEs from Frankia revealed their unexpected diversity and a vast array of predicted adaptive functions. Frankia ICEs were found to excise by site-specific recombination from their host's chromosome in vitro and in planta suggesting that they are functional mobile elements whether Frankiae live as soil saprophytes or plant endosymbionts. Phylogenetic analyses of proteins involved in ICEs maintenance and transfer suggests that active exchange between ICEs cargo-borne and chromosomal genes took place within the Actinomycetales order. Functionality of Frankia ICEs in vitro as well as in planta lets us anticipate that conjugation and ICEs could allow the development of genetic manipulation tools for this challenging microorganism and for many other Actinobacteria.     

The Resveratrol Tetramer (-)-Hopeaphenol Inhibits Type III Secretion in the Gram-Negative Pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa

Society faces huge challenges, as a large number of bacteria have developed resistance towards many or all of the antibiotics currently available. Novel strategies that can help solve this problem are urgently needed. One such strategy is to target bacterial virulence, the ability to cause disease e.g., by inhibition of type III secretion systems (T3SSs) utilized by many clinically relevant gram-negative pathogens. Many of the antibiotics used today originate from natural sources. In contrast, most virulence-blocking compounds towards the T3SS identified so far are small organic molecules. A recent high-throughput screening of a prefractionated natural product library identified the resveratrol tetramer (-)-hopeaphenol as an inhibitor of the T3SS in Yersinia pseudotuberculosis. In this study we have investigated the virulence blocking properties of (-)-hopeaphenol in three different gram-negative bacteria. (-)-Hopeaphenol was found to have micromolar activity towards the T3SSs in Yersinia pseudotuberculosis and Pseudomonas aeruginosa in cell-based infection models. In addition (-)-hopeaphenol reduced cell entry and subsequent intracellular growth of Chlamydia trachomatis.     

Functional Analysis of the Gonococcal Genetic Island of Neisseria gonorrhoeae

Neisseria gonorrhoeae is an obligate human pathogen that is responsible for the sexually-transmitted disease gonorrhea. N. gonorrhoeae encodes a T4SS within the Gonococcal Genetic Island (GGI), which secretes ssDNA directly into the external milieu. Type IV secretion systems (T4SSs) play a role in horizontal gene transfer and delivery of effector molecules into target cells. We demonstrate that GGI-like T4SSs are present in other β-proteobacteria, as well as in α- and γ-proteobacteria. Sequence comparison of GGI-like T4SSs reveals that the GGI-like T4SSs form a highly conserved unit that can be found located both on chromosomes and on plasmids. To better understand the mechanism of DNA secretion by N. gonorrhoeae, we performed mutagenesis of all genes encoded within the GGI, and studied the effects of these mutations on DNA secretion. We show that genes required for DNA secretion are encoded within the yaa-atlA and parA-parB regions, while genes encoded in the yfeB-exp1 region could be deleted without any effect on DNA secretion. Genes essential for DNA secretion are encoded within at least four different operons.     

Worldwide Occurrence of Integrative Conjugative Element Encoding Multidrug Resistance Determinants in Epidemic Vibrio cholerae O1

In the last decades, there has been an increase of cholera epidemics caused by multidrug resistant strains. Particularly, the integrative and conjugative element (ICE) seems to play a major role in the emergence of multidrug resistant Vibrio cholerae. This study fully characterized, by whole genome sequencing, new ICEs carried by multidrug resistant V. cholerae O1 strains from Nigeria (2010) (ICEVchNig1) and Nepal (1994) (ICEVchNep1). The gene content and gene order of these two ICEs are the same, and identical to ICEVchInd5, ICEVchBan5 and ICEVchHai1 previously identified in multidrug resistant V. cholerae O1. This ICE is characterized by dfrA1, sul2, strAB and floR antimicrobial resistance genes, and by unique gene content in HS4 and HS5 ICE regions. Screening for ICEs, in publicly available V. cholerae genomes, revealed the occurrence and widespread distribution of this ICE among V. cholerae O1. Metagenomic analysis found segments of this ICE in marine environments far from the direct influence of the cholera epidemic. Therefore, this study revealed the epidemiology of a spatio-temporal prevalent ICE in V. cholerae O1. Its occurrence and dispersion in V. cholerae O1 strains from different continents throughout more than two decades can be indicative of its role in the fitness of the current pandemic lineage.     

Missplicing suppressor alleles of Arabidopsis PRE-MRNA PROCESSING FACTOR 8 increase splicing fidelity by reducing the use of novel splice sites

Efficient splicing requires a balance between high-fidelity splice-site (SS) selection and speed. In Saccharomyces cerevisiae, Pre-mRNA processing factor 8 (Prp8) helps to balance precise SS selection and rapid, efficient intron excision and exon joining. argonaute1-52 (ago1-52) and incurvata13 (icu13) are hypomorphic alleles of the Arabidopsis thaliana genes ARGONAUTE1 (AGO1) and AUXIN RESISTANT6 (AXR6) that harbor point mutations creating a novel 3′SS and 5′SS, respectively. The spliceosome recognizes these novel SSs, as well as the intact genuine SSs, producing a mixture of wild-type and aberrant mature mRNAs. Here, we characterized five novel mutant alleles of PRP8 (one of the two Arabidopsis co-orthologs of yeast Prp8), naming these alleles morphology of ago1-52 suppressed5 (mas5). In the mas5-1 background, the spliceosome preferentially recognizes the intact genuine 3′SS of ago1-52 and 5′SS of icu13. Since point mutations that damage genuine SSs make the spliceosome prone to recognizing cryptic SSs, we also tested alleles of four genes carrying damaged genuine SSs, finding that mas5-1 did not suppress their missplicing. The mas5-1 and mas5-3 mutations represent a novel class of missplicing suppressors that increase splicing fidelity by hampering the use of novel SSs, but do not alter general pre-mRNA splicing.     

Biology and engineering of integrative and conjugative elements: Construction and analyses of hybrid ICEs reveal element functions that affect species-specific efficiencies

Integrative and conjugative elements (ICEs) are mobile genetic elements that reside in a bacterial host chromosome and are prominent drivers of bacterial evolution. They are also powerful tools for genetic analyses and engineering. Transfer of an ICE to a new host involves many steps, including excision from the chromosome, DNA processing and replication, transfer across the envelope of the donor and recipient, processing of the DNA, and eventual integration into the chromosome of the new host (now a stable transconjugant). Interactions between an ICE and its host throughout the life cycle likely influence the efficiencies of acquisition by new hosts. Here, we investigated how different functional modules of two ICEs, Tn916 and ICEBs1, affect the transfer efficiencies into different host bacteria. We constructed hybrid elements that utilize the high-efficiency regulatory and excision modules of ICEBs1 and the conjugation genes of Tn916. These elements produced more transconjugants than Tn916, likely due to an increase in the number of cells expressing element genes and a corresponding increase in excision. We also found that several Tn916 and ICEBs1 components can substitute for one another. Using B. subtilis donors and three Enterococcus species as recipients, we found that different hybrid elements were more readily acquired by some species than others, demonstrating species-specific interactions in steps of the ICE life cycle. This work demonstrates that hybrid elements utilizing the efficient regulatory functions of ICEBs1 can be built to enable efficient transfer into and engineering of a variety of other species.     

The role of type III secretion System 2 in Vibrio parahaemolyticus pathogenicity

Vibrio parahaemolyticus, a Gram-negative marine bacterial pathogen, is emerging as a major cause of food-borne illnesses worldwide due to the consumption of raw seafood leading to diseases including gastroenteritis, wound infection, and septicemia. The bacteria utilize toxins and type III secretion system (T3SS) to trigger virulence. T3SS is a multi-subunit needle-like apparatus used to deliver bacterial proteins, termed effectors, into the host cytoplasm which then target various eukaryotic signaling pathways. V. parahaemolyticus carries two T3SSs in each of its two chromosomes, named T3SS1 and T3SS2, both of which play crucial yet distinct roles during infection: T3SS1 causes cytotoxicity whereas T3SS2 is mainly associated with enterotoxicity. Each T3SS secretes a unique set of effectors that contribute to virulence by acting on different host targets and serving different functions. Emerging studies on T3SS2 of V. parahaemolyticus, reveal its regulation, translocation, discovery, characterization of its effectors, and development of animal models to understand the enterotoxicity. This review on recent findings for T3SS2 of V. parahaemolyticus highlights a novel mechanism of invasion that appears to be conserved by other marine bacteria.     

Conjugative Transfer and cis-Mobilization of a Genomic Island by an Integrative and Conjugative Element of Streptococcus agalactiae

Putative integrative and conjugative elements (ICEs), i.e., genomic islands which could excise, self-transfer by conjugation, and integrate into the chromosome of the bacterial host strain, were previously identified by in silico analysis in the sequenced genomes of Streptococcus agalactiae (M. Brochet et al., J. Bacteriol. 190:6913–6917, 2008). We investigated here the mobility of the elements integrated into the 3′ end of a tRNA^Lys gene. Three of the four putative ICEs tested were found to excise but only one (ICE_515_tRNA^Lys) was found to transfer by conjugation not only to S. agalactiae strains but also to a Streptococcus pyogenes strain. Transfer was observed even if recipient cell already carries a related resident ICE or a genomic island flanked by attL and attR recombination sites but devoid of conjugation or recombination genes (CIs-Mobilizable Element [CIME]). The incoming ICE preferentially integrates into the 3′ end of the tRNA^Lys gene (i.e., the attR site of the resident element), leading to a CIME-ICE structure. Transfer of the whole composite element CIME-ICE was obtained, showing that the CIME is mobilizable in cis by the ICE. Therefore, genomic islands carrying putative virulence genes but lacking the mobility gene can be mobilized by a related ICE after site-specific accretion.