Molecular Microbiology最新文献

BosR is a novel nucleic acid-binding protein in the ferric uptake regulator (FUR) family that regulates gene expression in the Lyme disease spirochete Borrelia (Borreliella) burgdorferi. This issue of Molecular Microbiology contains a comprehensive transcriptomic study that keenly defines the regulatory swath of BosR in the vertebrate host of B. burgdorferi. Despite homology to Fur-like and PurR-like orthologs, BosR has traditionally been linked to regulation of RpoS, the alternative sigma factor that controls the regulon required for establishing a vertebrate infection. However, BosR regulates other genes through an RpoS-independent mechanism, which is elegantly elaborated in Grassmann et al., along with clearly demonstrating that BosR does not participate in the defense against oxidative and nitrosative stress in the vertebrate. However, the recently recognized role of BosR as an RNA-binding protein with RNA chaperone activity that regulates gene expression in a post-transcriptional fashion is not wholly appreciated, which clouds the results on determining the DNA-binding site in vivo. Regardless, this seminal study enshrines BosR as a major regulator of gene expression in B. burgdorferi and delineates a multitude of BosR-regulated cellular functions in the spirochete related to its ability to navigate between its tick vector and vertebrate host in nature as well as to persist in these two disparate environments.

Aspergillus fumigatus is a ubiquitous filamentous fungus and dangerous human pathogen that produces a limited pool of small RNAs under standard laboratory conditions. To better understand the rules of small RNA production in A. fumigatus, we induced canonical RNA interference (RNAi) via overexpression of two separate inverted-repeat transgenes. We observed production of predominantly 20-nt, 5' uridine-containing small RNAs from the 3' end of each transgene nearest to the loop region and dependent on dicer-like B RNase III enzyme. Using this refined knowledge, we assessed small RNA biogenesis by sRNA-seq in three double knockout strains of the RNAi pathway, namely the dicer-like proteins (ΔdclA/B), argonautes (ΔppdA/B), and RNA-dependent RNA polymerases (ΔrrpA/B). In each case, we found limited evidence for production of 5' U-containing small RNAs reliant on the RNAi machinery under standard laboratory conditions. We did observe 5' U-containing small RNAs amongst the abundant tRNA-derived RNAs (tDRs); however, biogenesis of tDRs was predominantly Dicer-like independent. To more accurately define the complex tDR repertoire, we employed a cutting-edge tDR-sequencing approach that improved tRNA-half detection and revealed qualitative morphotype-specific changes in the small RNA fraction of conidia relative to mycelium. Finally, leveraging the limited sRNA repertoire of A. fumigatus, we tested the consequences of inverted-repeat transgene overexpression in the ΔdclA/B double knockout, which revealed growth inhibition even in the absence of double-stranded RNA (dsRNA) processing and small RNA production. We hypothesize that the RNAi substrate-limited landscape of A. fumigatus facilitates sensitivity to increases in dsRNA, offering an intriguing system for future studies of dsRNA metabolism.

Mycoplasma pneumoniae is a leading cause of bacterial community-acquired pneumonia, responsible for severe respiratory and extrapulmonary diseases in children and adults. The Community Acquired Respiratory Distress Syndrome (CARDS) toxin is a key virulence factor that exerts ADP-ribosylating and vacuolating activities on host cells, recapitulating the inflammatory and histopathological damage seen in infected airways. Although the host proteins annexin-A2 (AnxA2) and surfactant protein-A were previously identified as toxin receptors, their absence did not abrogate CARDS toxin activity. Intriguingly, our subsequent work identified an interaction between CARDS toxin and the ubiquitous membrane phospholipids sphingomyelin (SM) and phosphatidylcholine (PC). This study investigated whether CARDS toxin uses these lipids as functional cell surface receptors. Using enzyme-linked immunosorbent assays, we demonstrated that the carboxy region of CARDS toxin binds to SM and PC in a dose-dependent manner, exhibiting higher affinity for SM. Depletion of SM from airway epithelial cell surface significantly reduced CARDS toxin binding, internalization and retrograde transport, as shown by immunoblot, pull-down, immunofluorescence, and live-cell imaging. Furthermore, SM depletion markedly decreased toxin-induced vacuolation and its stability, a phenotype rescued by adding exogenous SM. Notably, combining SM depletion with AnxA2 suppression nearly abolished toxin binding, entry, and subsequent vacuolation. These findings establish that CARDS toxin utilizes SM as a functional receptor to mediate its activity, highlighting a critical lipid-dependent mechanism for host cell targeting.

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.

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 in vivo 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 in vivo for putative inhibitors of P. falciparum cytoadherence.