Aug 2026· Journal of Infectious Diseases· 0 citations
Medicine
TL;DR
Deletion of fabR reduced host cell adherence, increased serum sensitivity, enhanced IgG deposition, and decreased vitronectin binding compared with the wild-type strain, and ΔfabR mutants from multiple NTHi strains showed increased metabolic activity, indicating a conserved role for FabR in metabolic regulation.
Abstract
Background
Regulation of transcription is essential for the pathoadaptation of non-typeable Haemophilus influenzae(NTHi). We identified FabR as a previously unrecognized transcriptional regulator in NTHi through its binding to the promoter region of ompP5 and investigated its contribution to bacterial virulence and physiology.
Methods
A fabR deletion mutant (ΔfabR) was generated in NTHi strain 3655. The mutant was compared with the wild-type strain in assays of host cell adherence, serum resistance, immunoglobulin G (IgG) deposition, vitronectin binding, murine nasopharyngeal colonization, protein expression, membrane fatty acid composition, and metabolic activity. Metabolic activity was also evaluated in ΔfabR mutants generated in additional NTHi strains.
Results
Deletion of fabR reduced host cell adherence, increased serum sensitivity, enhanced IgG deposition, and decreased vitronectin binding compared with the wild-type strain. The ΔfabR mutant also exhibited impaired colonization of the murine nasopharynx. Proteomic analyses demonstrated altered expression of metabolic proteins, accompanied by an increased unsaturated-to-saturated fatty acid ratio. Furthermore, ΔfabR mutants from multiple NTHi strains showed increased metabolic activity, indicating a conserved role for FabR in metabolic regulation.
Conclusions
FabR is a novel transcriptional regulator that may contribute to NTHi virulence. By modulating bacterial metabolism and membrane lipid composition, FabR may influence outer membrane architecture, thereby affecting host interaction, immune evasion, and colonization.
The dynamic expression of UPEC's stress response mechanisms are emphasized and the central role of Bar/UvrY in modulating pathoadaptive mechanisms under antibiotic stress is outlined.
Ali Hazim Jabbar, M. Flayyih· Iraqi Journal of Science· 0 citations
Avian pathogenic Escherichia coli (APEC) induces avian colibacillosis and brings huge economic losses to global poultry production. The small alarmone (p)ppGpp mediates the bacterial stringent response, a vital pathway modulating microbial stress adaptation and pathogenic capacity. The functions of the (p)ppGpp synthase gene relA in APEC pathogenesis remain poorly characterized. In this study, we constructed a relA deletion mutant (ΔrelA) and its complemented strain (CΔrelA). The phenotypic and pathogenic characteristics of these strains were investigated. The results showed that deletion of relA did not significantly affect bacterial growth or motility. However, the ΔrelA strain showed increased susceptibility to aminoglycoside antibiotics. Furthermore, the enhanced interbacterial competition of the mutant was associated with the upregulation of core genes in the type VI secretion system (T6SS). Importantly, relA was essential for APEC adhesion to and invasion of avian DF-1 cells, as well as for colonization and virulence in ducklings, where ΔrelA exhibited significantly attenuated infectivity and reduced bacterial loads in the liver and spleen. Furthermore, transcriptomic analysis revealed that RelA deletion downregulated genes involved in integral components of the membrane, and further assays confirmed compromised membrane integrity in the mutant strain. These findings suggest that RelA maintains membrane integrity, which underpins its contributions to antibiotic resistance and virulence. These findings indicate that relA plays a key role in APEC virulence, antibiotic resistance, and membrane homeostasis, and could provide a theoretical basis for targeting the stringent response as a potential strategy to control avian colibacillosis.
Jiangang Hu, Dossêh Jean Apôtre Afayibo, Chang Liu et al.· Microorganisms· 0 citations
Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease, a chronic enteritis in ruminants, and is capable of persisting within macrophages despite the activation of host immune defenses. Although this intracellular persistence is a key determinant of MAP pathogenicity, the bacterial factors and host responses that regulate this process remain poorly understood. In this study, we established the first CRISPR interference (CRISPRi) platform applied to bovine monocyte-derived macrophages (MDM) to evaluate the functions of MAP genes involved in intracellular survival and to perform an integrative analysis of host transcriptomic responses. MAP mutants were targeted to two genes (mdh and MAP1981c). The optimal concentration of anhydrotetracycline (ATc) was determined to be 2 μg/ml by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to 72 h. The gene expression profiles and intracellular MAP levels were investigated using RNA-seq and colony-forming units, respectively. The survival rates of the MAP mutants significantly decreased with the time course of infection in MAP-mdhKD and MAP1981cKD (KD, knockdown). RNA-seq-based gene expression profiling suggested that target gene silencing in MAP mutants led to altered expression of host genes involved in lipid metabolism, T-cell activation reduction, and antimicrobial response in bovine MDM, contributing to reduced intracellular survival of MAP. Our study demonstrates that the downregulation of mdh and MAP1981c in MAP significantly alters the host transcriptomic landscape in bovine MDM, revealing their critical roles in subverting host immune defenses for intracellular persistence.
Jun Ho Lee, Eun-Seo Lee, Xi-Rui Xiang et al.· Microbial Pathogenesis· 1 citation
Avian pathogenic Escherichia coli (APEC) is a major cause of colibacillosis in poultry, yet the role of the ecpA gene, which encodes the major structural subunit of the Escherichia coli common pilus (ECP), remains incompletely defined in APEC pathogenesis. To investigate the role of ecpA in the biological characteristics and pathogenicity of Avian Pathogenic Escherichia coli (APEC) strain FJLY68, an ecpA deletion mutant (ΔecpA) and its corresponding complemented strain (CΔecpA) were constructed using the CRISPR/Cas9 system and verified by PCR and Sanger sequencing. Phenotypic analyses revealed that the ΔecpA mutation significantly impaired bacterial motility, biofilm formation, adherence to chicken embryonic fibroblast (DF-1) cells, and fimbriae assembly. Transcriptomic analysis identified 1720 differentially expressed genes in the ΔecpA mutant, significantly enriched in pathways associated with flagellar assembly, chemotaxis, and metabolism, consistent with the observed phenotypic changes. Although in vitro growth was unaffected, the ΔecpA mutant exhibited markedly attenuated virulence in a chick infection model, as indicated by an increased LD50, attenuated clinical signs and pathological lesions, and reduced bacterial colonisation in tissues. Full genetic complementation restored all observed defects to wild-type levels. This study identifies ecpA as a critical determinant of APEC pathogenesis, directly linking its function to bacterial motility, biofilm formation, adhesion, and in vivo virulence, and provides a theoretical basis for developing novel control strategies targeting this virulence factor.
ABSTRACT Haemophilus influenzae is a diverse human-restricted bacterium that normally colonizes the healthy nasopharynx but also causes common infections. Comparisons of clinical isolate genomes previously identified a gene, msf, that contained Sel1-like repeats that were associated with clinical disease. Mutant analysis had further found that msf improved survival in macrophages and increased systemic infection in an animal model. However, the role of msf in other conditions and its molecular function remain unknown. To identify protein-protein interactions with Msf, a yeast two-hybrid screen against an H. influenzae prey library was conducted, which found potential interactions with lipoprotein exporter protein LolD and an autotransporter adhesin Hap. To identify effects of msf on gene expression, we compared wild-type and mutant strains grown in multiple culture conditions by RNA-seq. The results indicate that msf modulates global gene expression in a condition-dependent manner, exerting an especially strong influence in starved surface-attached biofilm cells. The few consistent changes in mutants’ planktonic exponential and stationary phases included decreased expression of two paralogous autotransporter adhesins. By contrast, mutant cells in starved surface-attached biofilms had dramatic changes in expression, including upregulation of protein translation and downregulation of alternative carbon metabolism. However, assays of 24 hour biofilm phenotypes found only subtle gene expression changes. Together, the results point to a speculative model of Msf functioning as an envelope-associated chaperone whose presence affects the relative expression of proteins at the outer membrane. IMPORTANCE Comparing genomes from different clinical isolates of the same pathogenic bacterial species has identified genes associated with virulence, but many of these are understudied or have no known function. The msf gene was previously implicated as a virulence factor in Haemophilus influenzae, a common cause of mucosal diseases including middle-ear and chronic lung infections. This study finds that the msf gene causes condition-specific changes in gene expression, with especially dramatic changes in starved surface-attached biofilm cells. Along with identification of putative protein-protein interaction partners, the results provide new clues as to the molecular and cellular function of Msf, potentially as an envelope-associated chaperone involved in membrane protein trafficking. Understanding how virulence-associated genes like msf modulate bacterial responses to the environment may help explain why some bacterial strains remain harmless colonizers while others become pathogens. Comparing genomes from different clinical isolates of the same pathogenic bacterial species has identified genes associated with virulence, but many of these are understudied or have no known function. The msf gene was previously implicated as a virulence factor in Haemophilus influenzae, a common cause of mucosal diseases including middle-ear and chronic lung infections. This study finds that the msf gene causes condition-specific changes in gene expression, with especially dramatic changes in starved surface-attached biofilm cells. Along with identification of putative protein-protein interaction partners, the results provide new clues as to the molecular and cellular function of Msf, potentially as an envelope-associated chaperone involved in membrane protein trafficking. Understanding how virulence-associated genes like msf modulate bacterial responses to the environment may help explain why some bacterial strains remain harmless colonizers while others become pathogens.
Evangeline M Williams, Mary C. Marino, J. Hammond et al.· mBio· 0 citations
YgeP plays a central role in APEC pathogenesis by balancing two infection strategies: motility-mediated dissemination and colonization, and biofilm-mediated attachment and tolerance, and negatively regulates key virulence traits during the early stages of infection.
Weiyang Su, Zhe Li, Siqi Feng et al.· Veterinary Microbiology· 0 citations
