Gram-negative pathogens pose a significant threat due to their propensity for causing various infections, often coupled with formidable resistance to conventional antibiotic treatments. The development of antivirulence (AV) compounds emerges as a promising alternative strategy by disrupting virulence mechanisms rather than targeting bacterial viability. Aurodox has exhibited promising AV properties in previous studies by blocking the expression and function of the LEE-encoded type 3 secretion system (T3SS) in enterohaemorrhagic Escherichia coli , an injectosome that translocates effector proteins directly into host target cells. However, aurodox’s efficacy against the T3SS of other pathogens remained unanswered. Using quantitative real-time polymerase chain reaction, we show that aurodox exerts inhibitory effects on selected T3SS including those of Salmonella Typhimurium, Yersinia pseudotuberculosis and Vibrio parahaemolyticus . Imaging of RAW 264.7 cells infected with S . Typhimurium showed that aurodox protects against late stages of infection by blocking the expression of the SPI-2 T3SS. To elucidate a conserved mechanism of action, we compared transcriptomic datasets from both E. coli and S . Typhimurium treated with aurodox to identify orthologous genes differentially expressed in response to aurodox treatment across both pathogens. This study sheds light on potential mechanisms driving the action of this promising AV compound.

Environmental pollution by hard-to-degrade polymers is on a steep rise and impacting the entire ecosystem. Microbial keratinases are pivotal in the breakdown of protein polymers that are otherwise resistant to most proteases. In this study, we isolated and identified bacteria from Sua pan soil through morphological, biochemical, and 16SrRNA sequencing approaches and further assessed these isolates for their keratinase production potential. Among the screened isolates, four bacteria, Bacillus cereus Bac 2, Bacillus sp. Bac 1, Pseudomonas aeruginosa Bac 3, and Achromobacter insuavis Bac 4, exhibited the highest degradation activity. B. cereus Bac 2 produced the widest clearance zone, whereas A. insuavis Bac 4 produced the narrowest clearance zone on feather meal agar. Protein bands observed in SDS-PAGE gels for the selected isolates further supported the presence of keratinolytic enzymes. We also investigated the effect of temperature and pH on keratinase activity and determined that all the keratinases were alkaline proteases, with B. cereus Bac 2, P. aeruginosa Bac 3, and Bacillus sp. Bac 1 showing maximum activity at a pH of 8.5, while A. insuavis Bac 4 had maximal activity at a pH of 8.0. Overall, our results indicated that B. cereus Bac 2 keratinase had significantly higher activity across all temperature and pH ranges investigated, compared to all the other isolates (p ≤ 0.0001). These findings highlight the potential application of bacterial isolates from alkalophilic environments, in the eco-friendly degradation of feather waste, as valuable by-products such as organic fertilizers, peptides, and amino acids.

Attaching and effacing pathogens, including enterohemorrhagic Escherichia coli (EHEC), colonize their preferred intestinal niche by sensing diverse host-, diet-, and microbiota-derived signals and coordinating the expression of virulence factors. D-serine, a host metabolite abundant in urine but scarce in the intestine, restricts EHEC colonization by transcriptionally repressing the type 3 secretion system (T3SS) while activating the SOS stress response. However, the mechanism underlying virulence regulation by D-serine remains unestablished. Here, we show that multiple amino acids, including L-serine converge on this pathway, repressing the T3SS without inducing the SOS response. Transcriptomic analyses showed a common response to D- and L-serine dominated by repression of nitrogen stress response genes. Mutational analysis identified the response regulators NtrC and Nac as essential mediators of T3SS repression by both serine enantiomers. Disruption of L-serine deaminase enzymes crucially revealed that T3SS repression depends on cytoplasmic ammonia/ammonium release rather than sensing of intact serine. While EHEC lacks canonical D-serine catabolic capacity, through metabolomics we provide evidence of oxidative deamination activity, capable of producing this regulatory signal. Together, these findings establish a mechanistic link between amino acid catabolism, nitrogen stress signaling, and virulence regulation in EHEC, highlighting how metabolic flux fine-tunes pathogen adaptation to intestinal niches.

Gram-negative pathogens pose a significant threat due to their propensity for causing various infections, often coupled with formidable resistance to conventional antibiotic treatments. In light of this challenge, the development of antivirulence (AV) compounds emerges as a promising alternative strategy, aiming to disrupt key virulence mechanisms rather than directly targeting bacterial viability. One such compound, aurodox, derived from Streptomyces goldiniensis , has exhibited promising AV properties in our prior studies. Specifically, aurodox caused a marked downregulation in the expression and function of the E. coli type 3 secretion system (T3SS), a needle-like injectosome structure which is deployed to translocate effector proteins from the cytoplasm to the host target cells. However, the broader spectrum of aurodox’s efficacy against T3SS across diverse pathogens remained unanswered, prompting the focus of this work. Using quantitative real-time PCR, we show that aurodox exerts inhibitory effects on selected T3SS in various pathogens, including Salmonella typhimurium, Yersinia pseudotuberculosis , and Vibrio parahaemolyticus . However, aurodox was not a universal blocker of all secretion systems, showing selectivity in its mode-of-action, even within a single strain. This finding was verified using transcriptomics which demonstrated that aurodox selectively blocks the expression of the Salmonella typhimurium SPI-2 type T3SS whilst other pathogenicity islands, including the SPI-1 system were not inhibited. To delve deeper into the mechanisms governing aurodox’s efficacy against these pathogens, we analysed transcriptomic datasets from both E. coli and S. Typhimurium treated with aurodox. By identifying orthologous genes exhibiting differential expression in response to aurodox treatment across these pathogens, our study sheds light on the potential mechanisms underlying the action of this rediscovered antibiotic.

This study reassessed the medicinal potential of Harpagophytum procumbens and related taxa in Botswana using ethnobotanical, phytochemical, microbiological, and AI-assisted approaches. Field surveys revealed a broader than expected distribution of Harpagophytum across Botswana, and evidence of hybridisation between H. procumbens and H. zeyheri that contributes to chemotypic variability. In selected accessions, Microbiological assays demonstrated strong antimicrobial activity, particularly against resistant Klebsiella pneumoniae, while additional activity was observed against Escherichia coli and Pseudomonas aeruginosa. However, limited efficacy was recorded against methicillin-resistant Staphylococcus aureus and Mycobacterium tuberculosis. Phytochemical integration indicated pathway-dependent medicinal functions, with phenylethanoid glycosides associated with antioxidant activity and iridoid and terpenoid metabolites linked to anti-inflammatory and antimicrobial effects. Artificial intelligence-assisted synthesis established chemogeographic stratification across Botswana, and identified ecological zones with taxa that have distinct therapeutic potential. The findings highlighted Harpagophytum as a chemically heterogeneous but commercially valuable medicinal resource with potential applications in precision phytomedicine and targeted bioprospecting.