Transcriptional analysis revealed that compared to neutral conditions, osmotic stress-related genes were significantly upregulated in the ΔliaS strain under acidic conditions, indicating that LiaS modulates acid tolerance through transcriptional regulation.
Abstract
ABSTRACT LiaS is a histidine kinase receptor in two-component systems that senses environmental stress signals. This study investigated the roles of LiaS in Listeria monocytogenes stress resistance and virulence. Phenotypic assays showed that deletion of liaS (ΔliaS) resulted in significantly impaired growth under acidic, alkaline, and osmotic stresses, as well as reduced survival under strong acidic conditions. These defects were partially restored in the complemented strain (CΔliaS). The growth defects were also observed in the ΔliaS under Cu2+ or H2O2 exposure. Furthermore, the ΔliaS strain exhibited impaired invasion and intracellular migration in host cells, along with attenuated colonization in the liver and spleen at 24 h post-infection. Bacterial loads in the spleen of infected mice remained lower at 48 h. The mortality was also delayed in ΔliaS‑infected mice. Transcriptional analysis revealed that compared to neutral conditions, osmotic stress-related genes (except opucB and gbuC) were significantly upregulated in the ΔliaS strain under acidic conditions, indicating that LiaS modulates acid tolerance through transcriptional regulation. Collectively, LiaS is essential for the adaptation of Listeria monocytogenes to acidic, osmotic, and oxidative stresses. Its absence attenuates host cell invasion, intracellular motility, and organ-specific colonization, highlighting its dual role in environmental resilience and pathogenicity. These findings provide a theoretical basis for understanding host–pathogen interactions and offer new strategies against antimicrobial-resistant pathogens.
The biology of the pathogenic fungus
Aspergillus fumigatus
remains largely unexplored, in part due to the large number of hypothetical and uncharacterized proteins. In this study, we focused on the protein encoded by the Afu4g10610 gene, which is consistently up-regulated across multiple stress-related transcriptomic datasets, including both
in vitro
and
in vivo
infection models. Functional characterization through the generation of mutant strains revealed that deletion of Afu4g10610 compromises the response to cell wall stress induced by Congo Red and Calcofluor White, correlating with the downregulation of key cell wall integrity (CWI) pathway sensors (
wsc1
and
midA
). In addition, the mutant exhibits enhanced resistance to osmotic stress, consistent with altered expression of the high-osmolarity glycerol (HOG) pathway effectors
mpkC
and
sakA
. The deletion mutant also showed a moderate reduction in cytotoxicity toward A549 epithelial cells and altered TNF production in RAW 264.7 macrophages, whereas the overexpression strain exhibited a significant decrease in TNF levels. GRAsp analysis predicted this gene to be associated with the phenylalanine/tyrosine catabolic pathway. Accordingly, pyomelanin production and related metabolites were analyzed. Moreover, double deletion mutants ∆
maiA
∆
10610
and ∆
hmgA
led to a marked reduction in pyomelanin production, accompanied by altered tyrosine consumption and homogentisic acid (HGA) production. Since pyomelanin biosynthesis depends on the conversion of HGA into benzoquinone acetate (BQA), a step traditionally considered spontaneous, we investigated the potential interaction between Afu4g10610 and HGA. Molecular docking analysis supported the binding of HGA at the predicted dimer interface of the protein and suggested potential binding of FADH₂ to the protein, as an electron donor. Together, these findings identify Afu4g10610 as a stress-associated protein that contributes to cell wall and osmotic stress adaptation and suggest a potential contribution to HGA-to-BQA conversion during pyomelanin biosynthesis. More broadly, our results support the possible involvement of an enzymatic component in a step previously considered spontaneous in
A. fumigatus
.
Eduardo Pelegri-Martinez, U. Perez-Cuesta, Saioa Cendon-Sanchez et al.· Frontiers in Microbiology· 0 citations
ABSTRACT Listeria monocytogenes is a gram-positive facultative intracellular bacterium that causes listeriosis in humans and animals. Survival in diverse environments and host-associated niches requires coordinated regulation of metabolism, stress responses, and virulence. The LysR-type transcriptional regulator GltC is predicted to regulate glutamate metabolism, yet its broader contribution to metabolic regulation and pathogenicity in L. monocytogenes remains unclear. Here, we explored the role of GltC in nitrogen utilization, central carbon metabolism, stress tolerance, and virulence. Deletion of gltC had no detectable effect on growth under nutrient-rich, nitrogen-defined conditions or at acidic and alkaline pH, indicating that GltC is not required for basal fitness. Instead, loss of gltC triggered extensive transcriptional remodeling of nitrogen assimilation pathways, including altered expression of genes involved in glutamate synthesis, ammonium transport, and ethanolamine utilization, as well as condition-dependent changes in tricarboxylic acid cycle-associated genes. The ΔgltC mutant exhibited enhanced tolerance to oxidative stress, with improved growth and survival during hydrogen peroxide challenge and increased expression of oxidative stress defense and DNA repair genes. Deletion of gltC led to broad upregulation of the PrfA virulence regulon and a modest increase in hemolytic activity. The ΔgltC strain displayed significantly higher bacterial burdens in the spleen and liver in a murine infection model. Collectively, these results suggest that GltC contributes to the coordination of nitrogen metabolism and oxidative stress adaptation, while influencing bacterial fitness during systemic infection. IMPORTANCE Pathogenic bacteria must constantly balance nutrient acquisition, metabolic activity, and virulence to persist in changing environments. Listeria monocytogenes causes severe foodborne disease and must adapt to changing nutrient and stress conditions during infection. Understanding how metabolism influences virulence is critical for identifying new strategies to limit bacterial survival in the host. This study shows that GltC contributes to the coordination of nitrogen metabolism, oxidative stress adaptation, and bacterial fitness during L. monocytogenes infection. Although GltC is not required for basic growth, its loss alters nitrogen metabolic pathways, enhances oxidative stress tolerance, and is associated with increased bacterial burdens during murine infection. These findings provide new insights into how metabolic regulation influences bacterial adaptation and fitness during host infection. Pathogenic bacteria must constantly balance nutrient acquisition, metabolic activity, and virulence to persist in changing environments. Listeria monocytogenes causes severe foodborne disease and must adapt to changing nutrient and stress conditions during infection. Understanding how metabolism influences virulence is critical for identifying new strategies to limit bacterial survival in the host. This study shows that GltC contributes to the coordination of nitrogen metabolism, oxidative stress adaptation, and bacterial fitness during L. monocytogenes infection. Although GltC is not required for basic growth, its loss alters nitrogen metabolic pathways, enhances oxidative stress tolerance, and is associated with increased bacterial burdens during murine infection. These findings provide new insights into how metabolic regulation influences bacterial adaptation and fitness during host infection.
S. Ogunleye, Monzur Chowdhury, H. Abdelhamed· Microbiology spectrum· 0 citations
ClpB, an ATP-dependent molecular chaperone belonging to the Hsp100/Clp subfamily of AAA+ ATPases, plays a crucial role in protein disaggregation, thereby enhancing bacterial survival under stress conditions. Despite its well-conserved function in prokaryotes, the specific contributions of ClpB to the pathogenesis of the ruminant pathogen Mycoplasma bovis remain largely unexplored. In this study, we identified and functionally characterized a ClpB homolog in M. bovis. Biochemical assays confirmed that the recombinant ClpB protein exhibits intrinsic ATPase activity and, in cooperation with the DnaK chaperone system, efficiently mediates protein disaggregation in vitro. Through genome-wide transposon mutagenesis of the M. bovis HB0801 strain, we generated ClpB-deficient mutants that maintained normal growth kinetics and morphology at 37 °C but exhibited significant growth defects under thermal and oxidative stress conditions. Phenotypic analysis demonstrated that ClpB disruption attenuated key virulence traits, including impaired adhesion to host cells, marked reduction in biofilm formation, diminished pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) expression in BoMac cells. Furthermore, the reduced virulence of the ClpB mutant was investigated by DIA proteomic analyses, which revealed that the ClpB mutant strain altered distinct protein expression patterns related to proteostasis, including phosphotransferase system, serine-type peptidase activity, serine hydrolase activity, and chaperone-mediated protein folding that contribute to the stress response and virulence. These findings collectively demonstrate that ClpB serves as a multifunctional virulence determinant in M. bovis, orchestrating stress adaptation, host-pathogen interactions, and pathogenic potential through modulation of both protein quality control systems and virulence-associated pathways.
Hui Zhang, Doukun Lu, Gang Zhao et al.· International Journal of Bio...· 0 citations
This study provides a theoretical basis for elucidating the molecular mechanism by which YibT modulates salmonella pathogenicity and shows that yibT contributes to regulate adhesion and invasion of S. typhimurium, and its deletion attenuates bacterial virulence and host pathogenicity.
Bingbing Wang, Min Yue, Jianjun Chen et al.· Microbial Pathogenesis· 0 citations
Extracellular vesicles (EVs) serve as pivotal mediators of bacterial intercellular communication, facilitating survival under diverse environmental hostilities. However, the regulatory landscape of EVs under the complex interplay of antibiotic and heavy metal co-stress remains an enigma. Here, we integrated multi-omics analyses to decipher the impact of EVs derived from a multidrug-resistant Vibrio parahaemolyticus strain (VP38) on a susceptible strain (VP35) under separated or combined stress of carbenicillin (CARB) and copper (Cu). Unexpectedly, the EV-mediated effects were stress-type specific: they promoted survival under CARB stress but markedly reduced viability under Cu exposure. Mechanistically, transcriptomic analysis showed that EVs uptake was associated with transcriptional reprogramming in the recipient strain, including downregulation of copper efflux genes (e.g., cusB/R) and iron acquisition genes (e.g., TonB-dependent receptors). This expression pattern suggests a possible disturbance in intracellular ion homeostasis and may contribute to increased susceptibility to heavy metal stress. These findings support a context-dependent defense-burden model of EV-mediated stress adaptation under combined antibiotic and metal stress. This study not only advances our understanding of bacterial social behaviors but also highlights EV-induced metabolic interference as a potential therapeutic strategy against resistant pathogens in aquaculture.
Yan Yuan, Hongmin Zhang, Pengju Zhao et al.· Ecotoxicology and Environmen...· 0 citations
ABSTRACT ClpX functions as a component of the ClpXP protease, a conserved intracellular protease that regulates protein turnover, stress responses, and virulence in multiple bacterial species. Our lab has established that clpX is necessary for resistance to cell envelope targeting antibiotics, such as penicillin and daptomycin in Bacillus anthracis Sterne. Previous microarray data identified the msrA/B gene encoding a bifunctional methionine sulfoxide reductase as upregulated in the ΔclpX mutant. Methionine sulfoxide reductases (Msr) repair oxidatively damaged proteins by reducing methionine sulfoxide residues back to methionine. While Msr enzymes are primarily associated with oxidative stress, cell wall antibiotics induce expression of msrA1 and msrB in S. aureus. Here, we investigated the role of MsrA/B in oxidative and cell envelope stress. Our results show that although hydrogen peroxide and paraquat induce msrA/B expression, the ΔmsrA/B strain was not susceptible to either oxidant, whereas the ΔclpX strain was sensitive to both. We also found that loss of msrA/B conferred penicillin‐specific sensitivity, but, unlike ΔclpX, increased sensitivity was not seen with other cell wall or cell membrane targeting antibiotics. Inactivation of the catalytic cysteine of either Msr domain of MsrA/B failed to complement, suggesting that the reducing activity of MsrA/B is required for penicillin resistance. These findings indicate that while MsrA/B contributes to penicillin resistance, other proteins in the ClpXP modulon must also play a role in oxidative and cell envelope stress.
Aeron B Pennington, Salina Hona, Josey I Austin et al.· MicrobiologyOpen· 0 citations
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