Significance: YeiE has been identified as a master virulence factor ofCronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO₃²⁻). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

Macrophages produce itaconic acid in phagosomes in response to bacterial cell wall component lipopolysaccharide to eliminate invading pathogenic bacteria. Itaconic acid competitively inhibits the first enzyme of the bacterial glyoxylate cycle. To overcome itaconic acid stress, bacteria employ the bacterial LysR-type transcriptional regulator RipR. However, it remains unknown which molecule activates RipR in bacterial pathogenesis. In this study, we determined the crystal structure of the regulatory domain of RipR from the intracellular pathogen Salmonella. The RipR regulatory domain structure exhibited the typical dimeric arrangement with the putative ligand-binding site between the two subdomains. Our isothermal titration calorimetry experiments identified isocitrate as the physiological ligand of RipR, whose intracellular level is increased in response to itaconic acid stress. We further found that 3-phenylpropionic acid significantly decreased the resistance of the bacteria to an itaconic acid challenge. Consistently, the complex structure revealed that the compound is antagonistically bound to the RipR ligand-binding site. This study provides the molecular basis of bacterial survival in itaconic acid stress from our immune systems. Further studies are required to reveal biochemical activity, which would elucidate how Salmonella survives in macrophage phagosomes by defending against itaconic acid inhibition of bacterial metabolism.

Shiga toxin–producing Escherichia coli (STEC) infections pose a significant public health challenge, characterized by severe complications including hemolytic uremic syndrome (HUS) due to Shiga toxin (Stx) production. Current therapeutic approaches encounter a critical limitation, as conventional antibiotic treatment is contraindicated due to its propensity to trigger bacterial SOS response and subsequently enhance Stx production, which increases the likelihood of developing HUS in antibiotic-treated patients. The lack of effective, safe therapeutic options has created an urgent need for alternative treatment strategies for STEC infections. This study investigates the therapeutic potential of virulent bacteriophages (phages) against STEC O157:H7. Our findings demonstrate that these phages effectively reduce STEC populations to levels comparable to ciprofloxacin treatment, while crucially avoiding the induction of SOS response and subsequent enhancement of Stx production. This is a significant advantage over conventional antibiotics, which increase Stx levels. Furthermore, these phages exhibited broad-spectrum antimicrobial activity against multiple clinical STEC isolates without triggering toxin expression. The capability of virulent phages to effectively control STEC without enhancing toxin production represents a promising therapeutic strategy that combines antimicrobial efficacy with safety considerations. These characteristics indicate that virulent phages represent a potential solution to address the current therapeutic challenges in STEC infections, particularly in mitigating the risk of HUS development in infected patients.

The spiral shape of intestinal pathogen Campylobacter jejuni is critical for invasion of intestinal mucosa epithelial cells. Insofar as this cell morphology plays a role in the pathology of C. jejuni infection, its restructuring by pharmacological intervention could be an unexplored means to prevention of infection. We recently described that peptidoglycan hydrolase 3 (Pgp3) is involved in the spiral-shape formation of C. jejuni. We report herein the design and synthesis of the hydroxamate-based inhibitors targeting Pgp3. C. jejuni cells exposed to these inhibitors changed from the helical- to rod-shaped morphology, comparable to the case of the pgp3-deletion mutant. Evidence for the mechanism of action was provided by crystal structures of Pgp3 in complex with inhibitors, shedding light into the binding modes of inhibitors within the active site, supported by kinetics and molecular-dynamics simulations. C. jejuni exposed to these inhibitors underwent the morphological change from helical- to rod-shaped bacteria, an event that reduce the ability for invasion of the host cells. This proof of concept suggests that alteration of morphology affects the interference with the bacterial infection.