Comparative Genomics of Multiple Strains of Pseudomonas cannabina pv. alisalensis,a Potential Model Pathogen of Both Monocots and Dicots

Comparative genomics of closely related pathogens that differ in host range can provide insights into mechanisms of host-pathogen interactions and host adaptation. Furthermore, sequencing of multiple strains with the same host range reveals information concerning pathogen diversity and the molecular basis of virulence. Here we present a comparative analysis of draft genome sequences for four strains of Pseudomonas cannabina pathovar alisalensis (Pcal), which is pathogenic on a range of monocotyledonous and dicotyledonous plants. These draft genome sequences provide a foundation for understanding host range evolution across the monocot-dicot divide. Like other phytopathogenic pseudomonads, Pcal strains harboured a hrp/hrc gene cluster that codes for a type III secretion system. Phylogenetic analysis based on the hrp/hrc cluster genes/proteins, suggests localized recombination and functional divergence within the hrp/hrc cluster. Despite significant conservation of overall genetic content across Pcal genomes, comparison of type III effector repertoires reinforced previous molecular data suggesting the existence of two distinct lineages within this pathovar. Furthermore, all Pcal strains analyzed harbored two distinct genomic islands predicted to code for type VI secretion systems (T6SSs). While one of these systems was orthologous to known P. syringae T6SSs, the other more closely resembled a T6SS found within P. aeruginosa. In summary, our study provides a foundation to unravel Pcal adaptation to both monocot and dicot hosts and provides genetic insights into the mechanisms underlying pathogenicity.     

铜绿假单胞菌T6SS的组装、分泌、功能和调控

作为一种对抗真核细胞和原核细胞的强有力细菌武器,Ⅵ型分泌系统(type Ⅵ secretion system,T6SS)广泛存在于革兰氏阴性菌中。铜绿假单胞菌是一种对多种抗生素具有耐药性并能够在人体引发急性和慢性感染的条件致病菌,它编码3套独立的T6SS,分别为H1-、H2-和H3-T6SS。T6SS通过介导细菌间竞争、生物被膜的形成、金属离子的摄取以及与真核宿主细胞之间的相互作用,对铜绿假单胞菌在毒力和适应环境方面发挥重要作用。本文主要对铜绿假单胞菌T6SS的组装、效应蛋白的分泌、功能及调控机制展开综述,旨在为T6SS的研究提供一定的参考,并为铜绿假单胞菌感染的预防和治疗提供一定的指导。     

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.     

Characterization of Two Macrolide Resistance-Related Genes in Multidrug-Resistant Pseudomonas aeruginosa Isolates

In analyzing the drug resistance phenotype and mechanism of resistance to macrolide antibiotics of clinical Pseudomonas aeruginosa isolates, the agar dilution method was used to determine the minimum inhibitory concentrations (MICs), and PCR (polymerase chain reaction) was applied to screen for macrolide antibiotics resistance genes. The macrolide antibiotics resistance genes were cloned, and their functions were identified. Of the 13 antibiotics tested, P. aeruginosa strains showed high resistance rates (ranging from 69.5–82.1%), and MIC levels (MIC90 > 256 μg/ml) to macrolide antibiotics. Of the 131 known macrolide resistance genes, only two genes, mphE and msrE, were identified in 262 clinical P. aeruginosa isolates. Four strains (1.53%, 4/262) carried both the msrE and mphE genes, and an additional three strains (1.15%, 3/262) harbored the mphE gene alone. The cloned msrE and mphE genes conferred higher resistance levels to three second-generation macrolides compared to two first-generation ones. Analysis of MsrE and MphE protein polymorphisms revealed that they are highly conserved, with only 1–3 amino acids differences between the proteins of the same type. It can be concluded that even though the strains showed high resistance levels to macrolides, known macrolide resistance genes are seldom present in clinical P. aeruginosa strains, demonstrating that a mechanism other than this warranted by the mphE and msrE genes may play a more critical role in the bacteria’s resistance to macrolides.Key words: Pseudomonas aeruginosa, macrolide, resistance gene, mphE, msrE     

The archetype Pseudomonas aeruginosa proteins TssB and TagJ form a novel subcomplex in the bacterial type VI secretion system

In Pseudomonas aeruginosa three type VI secretion systems (T6SSs) coexist, called H1‐ to H3‐T6SSs. Several T6SS components are proposed to be part of a macromolecular complex resembling the bacteriophage tail. The T6SS protein HsiE1 (TagJ) is unique to the H1‐T6SS and absent from the H2‐ and H3‐T6SSs. We demonstrate that HsiE1 interacts with a predicted N‐terminal α‐helix in HsiB1 (TssB) thus forming a novel subcomplex of the T6SS. HsiB1 is homologous to the Vibrio cholerae VipA component, which contributes to the formation of a bacteriophage tail sheath‐like structure. We show that the interaction between HsiE1 and HsiB1 is specific and does not occur between HsiE1 and HsiB2. Proteins of the TssB family encoded in T6SS clusters lacking a gene encoding a TagJ‐like component are often devoid of the predicted N‐terminal helical region, which suggests co‐evolution. We observe that a synthetic peptide corresponding to the N‐terminal 20 amino acids of HsiB1 interacts with purified HsiE1 protein. This interaction is a common feature to other bacterial T6SSs that display a TagJ homologue as shown here with Serratia marcescens. We further show that hsiE1 is a non‐essential gene for the T6SS and suggest that HsiE1 may modulate incorporation of HsiB1 into the T6SS.     

Diversity of Pseudomonas Genomes,Including Populus-Associated Isolates,as Revealed by Comparative Genome Analysis

The Pseudomonas genus contains a metabolically versatile group of organisms that are known to occupy numerous ecological niches, including the rhizosphere and endosphere of many plants. Their diversity influences the phylogenetic diversity and heterogeneity of these communities. On the basis of average amino acid identity, comparative genome analysis of >1,000 Pseudomonas genomes, including 21 Pseudomonas strains isolated from the roots of native Populus deltoides (eastern cottonwood) trees resulted in consistent and robust genomic clusters with phylogenetic homogeneity. All Pseudomonas aeruginosa genomes clustered together, and these were clearly distinct from other Pseudomonas species groups on the basis of pangenome and core genome analyses. In contrast, the genomes of Pseudomonas fluorescens were organized into 20 distinct genomic clusters, representing enormous diversity and heterogeneity. Most of our 21 Populus-associated isolates formed three distinct subgroups within the major P. fluorescens group, supported by pathway profile analysis, while two isolates were more closely related to Pseudomonas chlororaphis and Pseudomonas putida. Genes specific to Populus-associated subgroups were identified. Genes specific to subgroup 1 include several sensory systems that act in two-component signal transduction, a TonB-dependent receptor, and a phosphorelay sensor. Genes specific to subgroup 2 contain hypothetical genes, and genes specific to subgroup 3 were annotated with hydrolase activity. This study justifies the need to sequence multiple isolates, especially from P. fluorescens, which displays the most genetic variation, in order to study functional capabilities from a pangenomic perspective. This information will prove useful when choosing Pseudomonas strains for use to promote growth and increase disease resistance in plants.     

Molecular Investigation of Outer Membrane Channel Genes Among Multidrug Resistance Clinical Pseudomonas Aeruginosa Isolates

Background:Multidrug resistance Pseudomonas aeruginosa (MDRPA) is most important issue in healthcare setting. It can secrete many virulence effector proteins via its secretion system type (T1SS-T6SS). They are using them as conductor for delivering the effector proteins outside to begins harmful effect on host cell increasing pathogenicity, competition against other microorganism and nutrient acquisition.Methods:The study include investigation of 50 isolates of MDRPA for transport secretion system and resistance for antibiotics. Molecular diagnosis using P. aeruginosa specific primer pairs, investigation of AprF, HasF, XcpQ, HxcQ, PscC, CdrB, CupB3, and Hcp using specific primer pairs by PCR were also performed.Results:The results revealed high resistance to beta lactam antibiotics (78% for ceftazidime, 78% for cefepime and 46% for piperacillin) can indicate possessing of isolates for beta lactamases and this confirmed by dropping resistance to piperacillin to 16% when combined with tazobactam. Also, the results shown the ability of MDRPA for pyocyanin biosynthesis using the system of genes.Conclusion:The current study conclude that all isolates of P. aeruginosa were highly virulent due to their possessing of all transport secretion system to deliver different effector proteins with possible harmful effects of these proteins.Key Words: Drug resistance, MDR, Efflux pump, Pseudomonas aeruginosa     

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.     

Fluorescent pseudomonads harboring type III secretion genes are enriched in the mycorrhizosphere of Medicago truncatula

Type III secretion systems (T3SSs) of Gram-negative bacteria mediate direct interactions with eukaryotic cells. Pseudomonas spp. harboring T3SS genes (T3SS+) were previously shown to be more abundant in the rhizosphere than in bulk soil. To discriminate the contribution of roots and associated arbuscular mycorrhizal fungi (AMF) on the enrichment of T3SS+ fluorescent pseudomonads in the rhizosphere of Medicago truncatula, their frequency was assessed among pseudomonads isolated from mycorrhizal and nonmycorrhizal roots and from bulk soil. T3SS genes were identified by PCR targeting a conserved hrcRST DNA fragment. Polymorphism of hrcRST in T3SS+ isolates was assessed by PCR-restriction fragment length polymorphism and sequencing. Genotypic diversity of all pseudomonads isolated, whether or not harboring T3SS, was described by BOX-PCR. T3SS+ pseudomonads were significantly more abundant in mycorrhizal than in nonmycorrhizal roots and in bulk soil, and all were shown to belong to the phylogenetic group of Pseudomonas fluorescens on the basis of 16S rRNA gene identity. Four hrcRST genotypes were described; two only included isolates from mycorrhizal roots. T3SS+ and T3SS- pseudomonads showed different genetic backgrounds as indicated by their different BOX-PCR types. Taken together, these data suggest that T3SSs are implicated in interactions between fluorescent pseudomonads and AM in medic rhizosphere.     

A Potent Anti-SpuE Antibody Allosterically Inhibits Type III Secretion System and Attenuates Virulence of Pseudomonas Aeruginosa

Multidrug-resistant gram-negative bacteria infection is particularly severe within the designated ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), which underscores the urgent need to explore alternative therapeutic strategies. The type III secretion system (T3SS) is considered to be a key virulence factor in many gram-negative bacteria, and T3SS is in turn regulated by SpuE in P. aeruginosa, which is a spermidine binding protein from an ATP-binding cassette transporter family and highly conserved within ESKAPE pathogens. Here, we identified a potent anti-SpuE antagonistic antibody that allosterically inhibits the expression of T3SS and attenuates virulence of P. aeruginosa. X-ray crystallography and molecular dynamics simulations revealed that binding of antibody to SpuE induces a change in the dynamics of SpuE, which in turn may reduce spermidine uptake by P. aeruginosa. The antibody could serve as a template for developing novel biologics to target a broad spectrum of gram-negative bacteria.     

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.     

Characterisation ofPseudomonas spp. isolated from foods

PutativePseudomonas spp. (102 isolates) from different foods were first characterised by API 20NE and then tested for some enzymatic activities (lipase and lecithinase production, starch hydrolysis and proteolytic activity). However subsequent molecular tests did not always confirm the results obtained, thus highlighting the limits of API 20NE. Instead RFLP ITS1 and the sequencing of 16S rRNA gene grouped the isolates into 6 clusters:Pseudomonas fluorescens (cluster I),Pseudomonas fragi (duster II and V)Pseudomonas migulae (cluster III),Pseudomonas aeruginosa (cluster IV) andPseudomonas chicorii (cluster VI). The pectinolytic activity was typical of species isolated from vegetable products, especiallyPseudomonas fluorescens. InsteadPseudomonas fragi, predominantly isolated from meat was characterised by proteolytic and lipolytic activities.     

Characterization of Exopolysaccharides Produced by Plant-Associated Fluorescent Pseudomonads

A total of 214 strains of plant-associated fluorescent pseudomonads were screened for the ability to produce the acidic exopolysaccharide (EPS) alginate on various solid media. The fluorescent pseudomonads studied were saprophytic, saprophytic with known biocontrol potential, or plant pathogenic. Approximately 10% of these strains exhibited mucoid growth under the conditions used. The EPSs produced by 20 strains were isolated, purified, and characterized. Of the 20 strains examined, 6 produced acetylated alginate as an acidic EPS. These strains included a Pseudomonas aeruginosa strain reported to cause a dry rot of onion, a strain of P. viridiflava with soft-rotting ability, and four strains of P. fluorescens. However, 12 strains of P. fluorescens produced a novel acidic EPS (marginalan) composed of glucose and galactose (1:1 molar ratio) substituted with pyruvate and succinate. Three of these strains were soft-rotting agents. Two additional soft-rotting strains of P. fluorescens produced a third acidic novel EPS composed of rhamnose, mannose, and glucose (1:1:1 molar ratio) substituted with pyruvate and acetate. When sucrose was present as the primary carbon source, certain strains produced the neutral polymer levan (a fructan) rather than an acidic EPS. Levan was produced by most strains capable of synthesizing alginate or the novel acidic EPS containing rhamnose, mannose, and glucose but not by strains capable of marginalan production. It is now evident that the group of bacteria belonging to the fluorescent pseudomonads is capable of elaborating a diverse array of acidic EPSs rather than solely alginate.     

Two copies of bla NDM-1 gene are present in NDM-1 producing Pseudomonas aeruginosa isolates from Serbia

New Delhi metallo-β-lactamase producing Pseudomonas aeruginosa isolates are of special interest since P. aeruginosa is a major cause of nosocomial infections, the treatment of which could now be jeopardized, especially in developing countries. Six additional NDM-1 positive P. aeruginosa clinical isolates belonging to two different genotypes were shown to be plasmid-free. PFGE-hybridization experiments revealed the chromosomal location of the bla _NDM-1 gene. Restriction analysis and hybridization revealed that two copies of the bla _NDM-1 gene are present in the genomes of all tested isolates, as in previously characterized P. aeruginosa MMA83. Moreover, it was shown that increasing imipenem concentration did not have the effect on copy number of the bla _NDM-1 gene in the genome of P. aeruginosa MMA83.