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Asp23-controlled cell wall imbalance promotes membrane vesicles biogenesis in Staphylococcus aureus

ABSTRACT: Membrane vesicles (MVs) are produced by species across all domains of life and have diverse physiological functions and promising applications. While the mechanisms for vesiculation in Gram-negative bacteria are well-established, the genetic determinant and regulation of MVs biogenesis in Gram-positive bacteria remain still largely unknown. Here, we demonstrate that a Q225P substitution in the alternative sigma factor SigB greatly triggers MVs production in Staphylococcus aureus strain Newman by directly repressing expression of alkaline shock protein 23, wherein the Q225P mutation hinders the specific binding of SigB to the asp23 promoter. Isogenic deletion of asp23 consistently promotes MVs formation in Newman, highlighting the critical role of both sigB and asp23 in modulating S. aureus vesiculation. While bacterial growth and cytoplasmic membrane fluidity do not be impaired, mutation of asp23 elicits a weakened cell wall and enhanced autolysis that potentially involved in LrgAB-controlled hydrolases and PSMα-mediated activities, which ultimately leads to hypervesiculation in Newman. Furthermore, TEM and proteomic analysis suggest nearly identical morphology and composition, but virulence-associated factors are significantly enriched in MVs upon asp23 deletion. Overall, this study reveals novel genetic determinants and cues underlying S. aureus vesiculation, advancing the understanding of the physiology of MVs biogenesis in Gram-positive bacteria.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Staphylococcus Aureus (strain Newman)

TISSUE(S): Blastoderm

SUBMITTER: Zhu Keting

LAB HEAD: Keting Zhu

PROVIDER: PXD048184 | Pride | 2024-01-10

REPOSITORIES: Pride

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Project description:We profiled the expression of circulating microRNAs (miRNAs) in mice exposed to gram-positive and gram-negative bacteria using Illumina small RNA deep sequencing. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) were used to induce bacterial infection in mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer. Following exposure to gram-negative bacteria alone, no differentially expressed miRNA was found in the injection, cut, or skin graft models. Exposure to mixed bacteria induced a similar expression pattern of the circulating miRNAs to that induced by gram-positive bacterial infection. Upon gram-positive bacterial infection, 9 miRNAs (mir-193b-3p, mir-133a-1-3p, mir-133a-2-3p, mir-133a-1-5p, mir-133b-3p, mir-434-3p, mir-127-3p, mir-676-3p, mir-215-5p) showed upregulation greater than 4-fold with a p-value < 0.01. Among them, mir-193b-3p, mir-133a-1-3p, and mir-133a-2-3p presented the most common miRNA targets expressed in the mice exposed to gram-positive bacterial infection. Male C57BL/6 mice (age, 10–12 weeks; weight, 30–35 g) were purchased from BioLasco (Yi-Lan, Taiwan). The mice were anesthetized by intraperitoneal injection of an anesthetic cocktail consisting of 0.1 mg/g ketamine and 0.01 mg/g xylazine. The anesthetized mice were restrained in a supine position on a heated pad to maintain body temperature at 37°C. Recombinant-specific gram-negative pathogen Escherichia coli (Xen14) and gram-positive pathogen Staphylococcus aureus (Xen29) purchased from Caliper (Caliper, USA) were used to induce bacterial infection in the mice at a concentration of 1 × 108 bacteria/100 μL of phosphate buffered saline (PBS). To create mixed gram-negative and gram-positive bacterial infection, 1 × 108 Xen14 bacteria and 1 × 108 Xen29 bacteria/100 μL of PBS were used for wound contamination. Three animal models were used to create bacterial infection routes: subcutaneous injection (hereafter referred to as (I)), cut wound (hereafter referred to as (C)), and skin grafting (hereafter referred to as (S)). In the (I) model, E. coli and/or S. aureus suspensions were injected subcutaneously into the backs of the mice using an Fr. 25 needle. In the (C) model, a 1 cm incision wound was created in the midline of the back, smeared with E. coli and/or S. aureus suspension, and the wound was closed directly with a 4-0 nylon suture. In the (S) model, a 1×1 cm rectangular full- thickness skin graft was lifted from the backs of the mice, E. coli and/or S. aureus suspensions were spread over the wound bed, and the skin graft was reattached and closed with a 4-0 nylon suture. An additional group of animals in each of these three models was inoculated with PBS to serve as a negative control. Small RNA libraries generated from the serum of mice after exposure to PBS, Xen14, Xen29, and Xen14+Xen29 via the routes of subcutaneous injection (I), cut wound (C), or under grafted skin (S) were analyzed using an Illumina HiSeq2000 Sequencer.

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Asp23-controlled cell wall imbalance promotes membrane vesicles biogenesis in Staphylococcus aureus

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