To establish infection, Salmonella confronts a dynamic barrage of host-induced stresses. The peptidoglycan layer is essential for maintaining bacterial cell integrity and counteracting these environmental stress. Its synthesis relies on the lipid carrier undecaprenyl phosphate, which is generated by the enzyme undecaprenyl pyrophosphate phosphatase (UppP). While UppP is linked to virulence in other pathogens, its role in Salmonella remains unclear. We show that an uppP mutant in S. Typhimurium exhibits altered cell morphology, reduced stiffness, and impaired survival in RAW 264.7 macrophages. The mutant is also attenuated in systemic infection in C57BL/6 mice. These defects are associated with increased sensitivity to nitrosative stress. Notably, iNOS inhibition or deficiency restores intracellular survival of the uppP mutant in both RAW 264.7 macrophages and the mouse model, implicating UppP in resistance to nitrosative stress. Our findings reveal a critical role for UppP in promoting Salmonella survival within macrophages and contributing to systemic pathogenesis.

We identified the RNA-binding protein Rop, encoded on the pOSAK1 plasmid of enterohaemorrhagic Escherichia coli (EHEC), as a novel factor that enhances nitric oxide (NO) resistance, although it has previously been reported to regulate plasmid copy number. The Rop-induced increase in NO resistance was significantly reduced in several small noncoding RNA (sRNA) gene-deficient EHEC mutants. Among these sRNAs, DsrA, ArcZ, and RprA were directly involved in the translational regulation of rpoS expression, suggesting that Rop modulates rpoS expression through sRNAs. To examine this mechanism, we generated sRNA gene-deficient mutants with an additional deletion of the 5' untranslated region (5' UTR) of rpoS, which is required for translational regulation. The increase in NO resistance by Rop was restored in the double mutant, suggesting that this phenotype is mediated by Rop-dependent interactions between sRNAs and the 5' UTR of rpoS mRNA. Furthermore, Rop promoted rpoS mRNA degradation, an effect that likely suppresses RpoS production and may thereby enhance NO resistance. Finally, an hfq-deficient EHEC mutant exhibited no increase in NO resistance in the presence of Rop, indicating that Hfq is essential for Rop-mediated NO resistance.

Many Gram-negative bacterial species use contact-dependent growth inhibition (CDI) systems to deliver toxic proteins into neighboring competitors. CDI+ strains deploy CdiA effector proteins, which translocate their C-terminal toxin (CT) domains into target bacteria through a receptor-mediated delivery pathway. To protect against auto-intoxication, CDI+ bacteria also produce CdiI immunity proteins that neutralize CT toxin activity. Here, we present the crystal structure of the CT·CdiIO32:H37 complex from Escherichia coli O32:H37. CTO32:H37 adopts the same fold as the tRNase domain of colicin D, and the nucleases share similar catalytic centers. However, unlike colicin D, which cleaves the anticodon loops of tRNAArg isoacceptors, CTO32:H37 exhibits nonspecific RNase activity. Notably, we find that endogenous elongation factor Tu (EF-Tu) co-purifies with the over-produced CT·CdiIO32:H37 complex. Although EF-Tu does not bind stably to CTO32:H37 in the absence of CdiIO32:H37, the translation factor is required for toxic RNase activity invitro. AlphaFold 3 modeling and site-directed mutagenesis indicate that CTO32:H37 interacts with the N-terminal GTPase domain of EF-Tu. EF-Tu appears to stabilize residue Trp52 within the hydrophobic core of the toxin, which in turn supports the RNase active site through an unusual hydrogen-bonding interaction with the catalytic His67 residue. Thus, EF-Tu is hijacked as an essential co-factor to organize the toxin's catalytic center.

The human malaria parasite Plasmodium falciparum invades red blood cells (RBCs) and exports parasite proteins to transform the host cell for its survival. These exported proteins facilitate cytoadherence of the infected RBC (iRBC) to endothelial cells of small blood vessels, protecting iRBCs from splenic clearance. The parasite protein PfEMP1 and the host protein CD36 play a major role in P. falciparum iRBC cytoadherence. The murine parasite Plasmodium berghei is a widely used experimental model that combines high genetic tractability with access to invivo studies. The P. berghei iRBC also sequesters by CD36-binding via an unknown parasite ligand and few parasite proteins, including EMAP1 and EMAP2, have been localised to the iRBC membrane. We have identified a new protein named EMAP3 and demonstrated its export to the iRBC membrane where it likely interacts with EMAP1, with only EMAP3 exposed on the outer surface of the iRBC. Parasites lacking EMAP3 display no significant reduction in growth or sequestration, indicating that EMAP3 is not a major CD36-binding protein. The outer-surface location of EMAP3 offers a new scaffold for displaying P. falciparum proteins on the surface of the P. berghei iRBC, providing a platform to screen invivo for putative inhibitors of P. falciparum cytoadherence.

The endomembrane system of the intestinal pathogen Giardia lamblia lacks a separate Golgi compartment. Without this sorting compartment, how cargo sorting to various subcellular destinations occurs within Giardia remains an open question. While the distribution of various Golgi-associated SNAREs and Rabs has been documented in this parasite, the TRAPP (TRAnsport Protein Particle) complex, a guanine nucleotide exchange factor for Golgi-associated Rabs, remained uncharacterized. Herein, we report that Giardia expresses a minimal set of TRAPP complex components, GlBet3, GlBet5, GlTrs23, and GlTrs31. Some of these components can interact with GlRab1a, GlRab11, and the COPII coat protein, GlSec23. Coupled with the colocalization and coimmunoprecipitation of GlBet3 and GlBet5, we propose the existence of a functional TRAPP complex in Giardia with an architecture that is different from that of yeast. While some interactions within this complex may be analogous to those in yeast, we find evidence of some unique interactions as well. The TRAPP genes are upregulated during encystation, and two components are associated with encystation-specific vesicles. Besides the endomembrane system, the presence of GlBet3 and GlBet5 at the plasma membrane, membrane wrapping ventral disc periphery, and the median body indicates that the TRAPP complex may support unique features and functions of Giardia.

ABSTRACTManno‐oligosaccharides and their metabolism play important roles in gut health, pharmaceutical development, and renewable chemical production. While the degradation of manno‐oligosaccharides has been previously studied, interest in bacterial mannobiose epimerases and mannoside phosphorylases is increasing because these enzymes provide a replacement for bacterial phosphotransferase systems as part of synthetic biology applications. In this report, we have physiologically and biochemically characterized the mannobiose epimerase and mannoside phosphorylase from the Gram‐negative saprophyte Cellvibrio japonicus, which is a bacterium that does not possess a phosphotransferase system for sugar import. While the initial stages of mannan degradation by C. japonicus have been studied, the physiological importance and biochemical activities of the mannosyl‐glucose phosphorylase (Mgp130A) and mannobiose epimerase (EpiA) predicted for latter stages of mannan metabolism were uncharacterized. After functional mutational analysis and biochemical assays of these two enzymes, we observed that both were essential for utilization of linear mannan, mannobiose, and mannotriose; however, only Mgp130A was critical for mannosyl‐glucose cleavage. A new plasmid (pJKN5) created during this study allowed for improved complementation analysis and uncovered a surprising toxic effect of galactose‐substituted manno‐oligosaccharides in strains lacking the epiA gene. Enzyme assay of Mgp130A revealed an enzyme with a high specific activity compared to other bacterial enzymes. Overall, this study advanced our understanding of C. japonicus mannan metabolism and contributed to the growing characterization of bacterial glycoside phosphorylases and epimerases important for biotechnology.

During one round of DNA replication, nearly 2000 ribonucleoside monophosphates (rNMPs) are incorporated in place of their cognate deoxyribonucleoside monophosphates (dNMPs). Given their high rate of insertion, genomic DNA could contain rNMPs that are damaged or mismatched. Here, we test the activity of Bacillus subtilis and Escherichia coli RNase HII on canonical, mismatched, and damaged rNMPs. We show that E. coli RNase HII is adept at incising most rNMP variants from DNA at similar frequencies, with the exception of an oxidized rNMP, where endoribonuclease activity is sharply reduced. In contrast, B. subtilis RNase HII efficiently incises rAMP, rCMP, and rUMP but is inefficient at processing rGMP in both a canonical and mismatched base pair. We test damaged ribonucleotides and find that B. subtilis RNase HII is refractory to processing abasic and oxidized ribonucleotide lesions. Our work shows that bacterial RNase HII enzymes have different intrinsic endoribonuclease activity toward the repair of canonical, mismatched, and damaged rNMPs, demonstrating that not all rNMP errors provoke efficient resolution. Our finding that B. subtilis RNase HII is recalcitrant to repairing damaged rNMPs resembles what is observed for eukaryotic RNase H2 orthologs, suggesting that other repair processes are necessary to resolve damaged rNMPs.

The treatment of Mycobacterium avium (Mav) infection, responsible for over 80% of nontuberculous mycobacterial pulmonary disease, remains challenging due to rising antibiotic resistance and unsatisfactory success rates. Hence, there is a need for a deeper understanding of host-pathogen interactions to inform the development of alternative therapeutic approaches, like host-directed therapy (HDT), aimed at improving host antimycobacterial defenses. However, compared to Mycobacterium tuberculosis (Mtb) infections, knowledge of host-pathogen interactions for Mav infection is still limited. To address this knowledge gap, we performed a genome-wide host transcriptomic analysis of Mav-infected primary human macrophages-the primary host cell-alongside Mtb-infected macrophages to leverage insights from Mtb research. Our findings show substantial overlap in the gene expression patterns between Mav-infected and Mtb-infected macrophages, including induction of cytokine responses and modulation of various G-protein coupled receptors (GPCRs) involved in (lipid-mediated) macrophage immune functions. Notable differences were observed in the expression of immediate early genes (IEGs), phospholipases, and genes of the GTPase of immunity-associated protein (GIMAP) family. This study laid a foundation for identifying both shared and Mav-specific host response pathways, providing direction for future investigations into host-pathogen interactions during Mav infection and the identification of novel targets for HDT.