Biology subjects

Dillard, J. P.

Publications and source records attributed to Dillard, J. P..

3 recordsLinked to original sources

bioRxiv2021

Listeria monocytogenes GlmR is an accessory uridyltransferase essential for cytosolic survival and virulence

The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions for these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell wall stress, cytosolic survival, inflammasome avoidance and ultimately virulence in vivo. A genetic suppressor screen revealed that blocking utilization of UDP-GlcNAc by a non-essential wall teichoic acid decoration pathway restored resistance to cell wall stress and partially restored virulence of {Delta}glmR mutants. In parallel, metabolomics revealed that {Delta}glmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggest that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell wall stress responses and virulence in vivo. Taken together these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes. ImportanceBacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK) to survive in the host cytosol. GlmR is important for resistance to some cell wall stresses and is essential for virulence. The {Delta}glmR mutant is deficient in production of an essential cell wall metabolite, UDP-GlcNAc, and suppressors which increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc and this enzymatic activity is conserved in pathogens from Firmicutes and Actinobacteria phyla. These results highlight the importance accessory cell wall metabolism enzymes in responding to cell wall stress in a variety of bacterial pathogens.

microbiology↗

bioRxiv2021

The AmiC/NlpD pathway dominates peptidoglycan breakdown in Neisseria meningitidisand affects cell separation, NOD1 agonist production, and infection

The human-restricted pathogen Neisseria meningitidis, which is best known for causing invasive meningococcal disease, has a nonpathogenic lifestyle as an asymptomatic colonizer of the human naso- and oropharyngeal space. N. meningitidis releases small peptidoglycan (PG) fragments during growth. It was demonstrated previously that N. meningitidis releases low levels of tripeptide PG monomer, which is an inflammatory molecule recognized by the human intracellular innate immune receptor NOD1. In this present study, we demonstrated that N. meningitidis released more PG-derived peptides compared to PG monomers. Using a reporter cell line overexpressing human NOD1, we showed that N. meningitidis activates NOD1 using PG-derived peptides. Generation of such peptides required the presence of the periplasmic N-acetylmuramyl-L-alanine amidase AmiC, and the outer membrane lipoprotein, NlpD. AmiC and NlpD were found to function in cell separation, and mutation of either amiC or nlpD resulted in large clumps of unseparated N. meningitidis cells instead of the characteristic diplococci. Using stochastic optical reconstruction microscopy, we demonstrated that FLAG epitope-tagged NlpD localized to the septum, while similarly-tagged AmiC was found at the septum in some diplococci but distributed around the cell in most cases. In a human whole blood infection assay, an nlpD mutant was severely attenuated and showed particular sensitivity to complement. Thus, in N. meningitidis the cell separation proteins AmiC and NlpD are necessary for NOD1 stimulation and for survival during infection of human blood.

microbiology↗

bioRxiv2021

Transcriptional and translational responsiveness of the Neisseria gonorrhoeae type IV secretion system to conditions of host infections

The type IV secretion system of Neisseria gonorrhoeae translocates single-stranded DNA into the extracellular space, facilitating horizontal gene transfer and initiating biofilm formation. Expression of this system has been observed to be low under laboratory conditions, and multiple levels of regulation have been identified. We used a translational fusion of lacZ to traD, the gene for the type IV secretion system coupling protein, to screen for increased type IV secretion system expression. We identified several physiologically relevant conditions, including surface adherence, decreased manganese or iron, and increased zinc or copper, which increase gonococcal type IV secretion system protein levels through transcriptional and/or translational mechanisms. These metal treatments are reminiscent of the conditions in the macrophage phagosome. The ferric uptake regulator, Fur, was found to repress traD transcript levels, but to also have a second role, acting to allow TraD protein levels to increase only in the absence of iron. To better understand type IV secretion system regulation during infection, we examined transcriptomic data from active urethral infection samples from five men. These data demonstrated differential expression of 20 of 21 type IV secretion system genes during infection, indicating upregulation of genes necessary for DNA secretion during host infection.

microbiology↗