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The lytic transglycosylase, LtgG, controls cell morphology and virulence in Burkholderia pseudomallei.

ABSTRACT: Burkholderia pseudomallei is the causative agent of the tropical disease melioidosis. Its genome encodes an arsenal of virulence factors that allow it, when required, to switch from a soil dwelling bacterium to a deadly intracellular pathogen. With a high intrinsic resistance to antibiotics and the ability to overcome challenges from the host immune system, there is an increasing requirement for new antibiotics and a greater understanding into the molecular mechanisms of B. pseudomallei virulence and dormancy. The peptidoglycan remodeling enzymes, lytic transglycosylases (Ltgs) are potential targets for such new antibiotics. Ltgs cleave the glycosidic bonds within bacterial peptidoglycan allowing for the insertion of peptidoglycan precursors during cell growth and division, and cell membrane spanning structures such as flagella and secretion systems. Using bioinformatic analysis we have identified 8 putative Ltgs in B. pseudomallei K96243. We aimed to investigate one of these Ltgs, LtgG (BPSL3046) through the generation of deletion mutants and biochemical analysis. We have shown that LtgG is a key contributor to cellular morphology, division, motility and virulence in BALB/c mice. We have determined the crystal structure of LtgG and have identified various amino acids likely to be important in peptidoglycan binding and catalytic activity. Recombinant protein assays and complementation studies using LtgG containing a site directed mutation in aspartate 343, confirmed the essentiality of this amino acid in the function of LtgG.

SUBMITTER: Jenkins CH

PROVIDER: S-EPMC6667503 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

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The lytic transglycosylase, LtgG, controls cell morphology and virulence in Burkholderia pseudomallei.

Jenkins Christopher H CH Wallis Russell R Allcock Natalie N Barnes Kay B KB Richards Mark I MI Auty Joss M JM Galyov Edouard E EE Harding Sarah V SV Mukamolova Galina V GV

Scientific reports 20190730 1

Burkholderia pseudomallei is the causative agent of the tropical disease melioidosis. Its genome encodes an arsenal of virulence factors that allow it, when required, to switch from a soil dwelling bacterium to a deadly intracellular pathogen. With a high intrinsic resistance to antibiotics and the ability to overcome challenges from the host immune system, there is an increasing requirement for new antibiotics and a greater understanding into the molecular mechanisms of B. pseudomallei virulenc ...[more]

PMID: 31363151

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Project description:BackgroundBurkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics.MethodsThe expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared.ResultsProteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative β-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in β-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified.ConclusionsAnalyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.

Project description:BackgroundBurkholderia pseudomallei is a tropical pathogen that causes melioidosis. Its intrinsic drug-resistance is a leading cause of treatment failure, and the few available antibiotics require prolonged use to be effective. This study aimed to assess the clinical potential of B. pseudomallei phages isolated from Hainan, China.MethodsBurkholderia pseudomallei strain (HNBP001) was used as the isolation host, and phages were recovered from domestic environmental sources, which were submitted to the host range determination, lytic property assays, and stability tests. The best candidate was examined via the transmission electron microscope for classification. With its genome sequenced and analyzed, its protective efficacy against B. pseudomallei infection in A549 cells and Caenorhabditis elegans was evaluated, in which cell viability and survival rates were compared using the one-way ANOVA method and the log-rank test.ResultsA phage able to lyse 24/25 clinical isolates was recovered. It was classified in the Podoviridae family and was found to be amenable to propagation. Under the optimal multiplicity of infection (MOI) of 0.1, an eclipse period of around 20 min and a high titer (1012 PFU/ml) produced within 1 h were demonstrated. This phage was found stabile at a wide range of temperatures (24, 37, 40, 50, and 60 °C) and pH values (3-12). After being designated as vB_BpP_HN01, it was fully sequenced, and the 71,398 bp linear genome, containing 93 open reading frames and a tRNA-Asn, displayed a low sequence similarity with known viruses. Additionally, protective effects of applications of vB_BpP_HN01 (MOI = 0.1 and MOI = 1) alone or in combination with antibiotics were found to improve viability of infected cells (70.6 ± 6.8%, 85.8 ± 5.7%, 91.9 ± 1.8%, and 96.8 ± 1.8%, respectively). A significantly reduced mortality (10%) and a decreased pathogen load were demonstrated in infected C. elegans following the addition of this phage.ConclusionsAs the first B. pseudomallei phage was isolated in Hainan, China, phage vB_BpP_HN01 was characterized by promising lytic property, stability, and efficiency of bacterial elimination during the in vitro/vivo experiments. Therefore, we can conclude that it is a potential alternative agent for combating melioidosis.

Project description:Infection by the Gram-negative pathogen Burkholderia pseudomallei results in the disease melioidosis, acquired from the environment in parts of southeast Asia and northern Australia. Clinical symptoms of melioidosis range from acute (fever, pneumonia, septicemia, and localized infection) to chronic (abscesses in various organs and tissues, most commonly occurring in the lungs, liver, spleen, kidney, prostate and skeletal muscle), and persistent infections in humans are difficult to cure. Understanding the basic biology and genomics of B. pseudomallei is imperative for the development of new vaccines and therapeutic interventions. This formidable task is becoming more tractable due to the increasing number of B. pseudomallei genomes that are being sequenced and compared. Here, we compared three B. pseudomallei genomes, from strains MSHR668, K96243 and 1106a, to identify features that might explain why MSHR668 is more virulent than K96243 and 1106a in a mouse model of B. pseudomallei infection. Our analyses focused on metabolic, virulence and regulatory genes that were present in MSHR668 but absent from both K96243 and 1106a. We also noted features present in K96243 and 1106a but absent from MSHR668, and identified genomic differences that may contribute to variations in virulence noted among the three B. pseudomallei isolates. While this work contributes to our understanding of B. pseudomallei genomics, more detailed experiments are necessary to characterize the relevance of specific genomic features to B. pseudomallei metabolism and virulence. Functional analyses of metabolic networks, virulence and regulation shows promise for examining the effects of B. pseudomallei on host cell metabolism and will lay a foundation for future prediction of the virulence of emerging strains. Continued emphasis in this area will be critical for protection against melioidosis, as a better understanding of what constitutes a fully virulent Burkholderia isolate may provide for better diagnostic and medical countermeasure strategies.

Dataset Information

The lytic transglycosylase, LtgG, controls cell morphology and virulence in Burkholderia pseudomallei.

Publications

The lytic transglycosylase, LtgG, controls cell morphology and virulence in Burkholderia pseudomallei.

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