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TatD DNases Contribute to Biofilm Formation and Virulence in Trueperella pyogenes.

ABSTRACT: TatD DNases are conserved proteins in a variety of organisms and are considered potential virulence factors in Plasmodium falciparum and Streptococcus pneumoniae. However, the function of TatD DNases has not yet been determined in Trueperella pyogenes, which causes various infections in animals and leads to economic losses. In this study, we describe the roles of TatD DNases in T. pyogenes (TpTatDs). A bioinformatics analysis was performed to investigate the sequence characteristics of TpTatDs, and then the ability of recombinant TatD proteins to hydrolyze DNA was determined in the presence of divalent cations. Moreover, we constructed tatD-deficient mutants. The biofilms formed by the wild-type and mutant strains were observed under a microscope. The mortality and bacterial load in the spleen of mice infected with the wild-type strain and tatD-deficient mutants were determined to obtain insights into the role of TatDs in the virulence of T. pyogenes. Two TatD DNases were identified in T. pyogenes. They were Mg²⁺-dependent DNases and exhibited DNA endonuclease activity. Compared with those formed by the parental strain, biofilms formed by mutants showed a significantly reduced thickness and biomass. Moreover, mutants produced a lower bacterial load in the spleen of mice and compromised virulence. Our data indicated that TatD DNases in T. pyogenes are involved in biofilm formation and required for virulence during infections.

SUBMITTER: Zhang Z

PROVIDER: S-EPMC8634637 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Biofilms play an important role in the pathogenesis of group A streptococcus (GAS), a Gram-positive pathogen responsible for a wide range of infections and with a significant public health impact. Although most GAS serotypes are able to form biofilms, there is a large amount of heterogeneity between individual strains in biofilm formation, as measured by standard crystal violet assays. It is generally accepted that biofilm formation includes the initial adhesion of bacterial cells to a surface followed by microcolony formation, biofilm maturation, and extensive production of extracellular matrix that links together proliferating cells and provides a scaffold for the three-dimensional (3D) biofilm structure. However, our studies show that for GAS strain JS95, microcolony formation is not an essential step in static biofilm formation, and instead, biofilm can be effectively formed from slow-growing or nonreplicating late-exponential- or early-stationary-phase planktonic cells via sedimentation and fixation of GAS chains. In addition, we show that the GAS capsule specifically contributes to the alternative sedimentation-initiated biofilms. Microcolony-independent sedimentation biofilms are similar in morphology and 3D structure to biofilms initiated by actively dividing planktonic bacteria. We conclude that GAS can form biofilms by an alternate noncanonical mechanism that does not require transition from microcolony formation to biofilm maturation and which may be obscured by biofilm phenotypes that arise via the classical biofilm maturation processes.IMPORTANCE The static biofilm assay is a common tool for easy biomass quantification of biofilm-forming bacteria. However, Streptococcus pyogenes biofilm formation as measured by the static assay is strain dependent and yields heterogeneous results for different strains of the same serotype. In this study, we show that two independent mechanisms, for which the protective capsule contributes opposing functions, may contribute to static biofilm formation. We propose that separation of these mechanisms for biofilm formation might uncover previously unappreciated biofilm phenotypes that may otherwise be masked in the classic static assay.

Project description:Trueperella pyogenes (T. pyogenes) is an important opportunistic animal pathogen that causes huge economic losses to the animal husbandry industry. The emergence of bacterial resistance and the unsatisfactory effect of the vaccine have prompted investigators to explore alternative strategies for controlling T. pyogenes infection. Due to the ability of phages to kill multidrug-resistant bacteria, the use of phage therapy to combat multidrug-resistant bacterial infections has attracted attention. In this study, a T. pyogenes phage, vB-ApyS-JF1 (JF1), was isolated from sewage samples, and its whole genome and biological characteristics were elucidated. Moreover, the protective effect of phage JF1 on a mouse bacteremic model caused by T. pyogenes was studied. JF1 harbors a double-stranded DNA genome with a length of 90,130 bp (30.57% G + C). The genome of JF1 lacked bacterial virulence-, antibiotic resistance- and lysogenesis-related genes. Moreover, the genome sequence of JF1 exhibited low coverage (<6%) with all published phages in the NCBI database, and a phylogenetic analysis of the terminase large subunits and capsid indicated that JF1 was evolutionarily distinct from known phages. In addition, JF1 was stable over a wide range of pH values (3 to 11) and temperatures (4 to 50°C) and exhibited strong lytic activity against T. pyogenes in vitro. In murine experiments, a single intraperitoneal administration of JF1 30 min post-inoculation provided 100% protection for mice against T. pyogenes infection. Compared to the phosphate-buffered saline (PBS) treatment group, JF1 significantly (P < 0.01) reduced the bacterial load in the blood and tissues of infected mice. Meanwhile, treatment with phage JF1 relieved the pathological symptoms observed in each tissue. Furthermore, the levels of the inflammatory cytokines tumour necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and interleukin-6 (IL-6) in the blood of infected mice were significantly (P < 0.01) decreased in the phage-treated group. Taken together, these results indicate that phage JF1 demonstrated great potential as an alternative therapeutic treatment against T. pyogenes infection.

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TatD DNases Contribute to Biofilm Formation and Virulence in Trueperella pyogenes.

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