Biology subjects

McPhee, J. B.

Publications and source records attributed to McPhee, J. B..

5 recordsLinked to original sources

bioRxiv2026

Division-arrest induced filamentation protects uropathogenic Escherichia coli from killing by the cathelicidin antimicrobial peptide LL-37

Antimicrobial peptides are a key component of the innate immune system, serving both as signaling and immunomodulatory molecules and also as direct antimicrobials toward numerous bacterial species. One of the key antimicrobial peptides in humans is LL-37 but the mechanism of bacterial killing remains poorly characterized. Using live-cell imaging, we observe that LL-37 preferentially targets actively dividing E. coli cells. Permeabilization occurs during late-stage division at the bacterial septum with both of the newly dividing cells being killed at the same time. Death occurs downstream of the recruitment and dissembly of FtsZ and FtsN and concomitantly with AmiB association at the divisome, consistent with killing occurring during late division. Model membrane tubes that topologically mimic the late-stage cytoplasmic membrane intermediates during division also undergo rapid size- and lipid composition-dependent fission, which likely reflect the ability of LL-37 to disrupt membranes. Intriguingly, we show that inducing bacterial filamentation results in resistance to LL-37 mediated killing. Resistance to LL-37 appears to be a general feature of bacterial filamentation as overexpression of queE, sulA or damX all resulted in reduced killing by LL-37. As filamentation is often observed during UPEC infection, we hypothesize that filamentation associated resistance to host innate immunity may be a key feature of UPEC pathogenesis that protects bacteria during extrusion from infection bladder epithelial cells, when exposure to soluble and cellular host defences is likely highest. This observation may provide an important avenue for therapeutic intervention. SignificanceAntimicrobial peptides are a key component of innate immunity. The human cathelicidin peptide, LL-37, is membrane active but the mechanism of sensitization in living systems is poorly understood. We show that dividing E. coli cells are highly susceptible to LL-37 mediated disruption. Killing occurs downstream of the assembly and activation of functional divisomes. In vitro membrane nanotubes also demonstrate sensitivity to LL-37 in a lipid composition and diameter-dependent manner. Cells with stalled division are protected from LL-37. Urinary tract infections caused by uropathogenic E. coli (UPEC) are a major cause of morbidity globally. UPEC form filamentous bacteria late in the human infection cycle. UPEC filaments are protected from LL-37 killing and suggest that interfering with bacterial filamentation might potentiate innate immune control of UTIs.

microbiology↗

bioRxiv2022

Salmonella actively modulates TFEB in murine macrophages in a growth-phase and time-dependent manner

The transcription factor TFEB drives expression of lysosomal, autophagic, and immune-responsive genes in response to LPS and phagocytosis. Interestingly, compounds that promote TFEB activity enhance bactericidal activity while intracellular pathogens like Mycobacterium and Salmonella repress TFEB. However, Salmonella enterica sv. Typhimurium (S. Typhimurium) was reported to actively stimulate TFEB, implying a benefit to Salmonella. To better understand the relationship between S. Typhimurium and TFEB, we assessed if S. Typhimurium regulated TFEB in macrophages in a manner dependent on infection conditions. We observed that macrophages that engulfed late-logarithmic grown Salmonella accumulated nuclear TFEB, comparable to macrophages that engulfed E. coli. In contrast, stationary-phase S. Typhimurium infection of macrophages actively delayed TFEB nuclear mobilization. The delay in TFEB nuclear mobilization was not observed in macrophages that engulfed heat-killed stationary-phase Salmonella, or Salmonella lacking functional SPI-1 and SPI-2 type three secretion systems. S. Typhimurium mutated in the master virulence regulator phoP or the secreted effector genes sifA, and sopD also showed TFEB nuclear translocation. Interestingly, while E. coli survived better in tfeb-/- macrophages, S. Typhimurium growth was similar in wild-type and tfeb-/- macrophages. Moreover, Salmonella survival was not readily affected by its growth phase in wild-type or knockout macrophages, though in HeLa cells late-log Salmonella benefitted from the loss of TFEB. Priming macrophages with phagocytosis enhanced the killing of Salmonella in wild-type, but not in tfeb-/- macrophages. Collectively, S. Typhimurium orchestrate TFEB in a manner dependent on infection conditions, while disturbing this context-dependent control of TFEB may be detrimental to Salmonella survival. ImportanceActivation of the host transcription factor TFEB helps mammalian cells adapt to stresses such as starvation and infection by upregulating lysosome, autophagy, and immuno-protective gene expression. Thus, TFEB is generally thought to protect host cells. However, it may also be that pathogenic bacteria like Salmonella orchestrate TFEB in a spatio-temporal manner to harness its functions to grow intracellularly. Indeed, the relationship between Salmonella and TFEB is controversial since some studies showed that Salmonella actively promotes TFEB, while others have observed that Salmonella degrades TFEB and that compounds that promote TFEB restrict bacterial growth. Our work provides a path to resolve these apparent discordant observations since we showed that stationary-grown Salmonella actively delays TFEB after infection, while late-log Salmonella is permissive of TFEB activation. Nevertheless, the exact function of this manipulation remains unclear, but conditions that erase the conditional control of TFEB by Salmonella may be detrimental to the microbe.

cell biology↗

bioRxiv2021

A mutation in the pmrD gene of Shigella flexneri abrogates functional PhoPQ-PmrD-PmrAB signaling and polymyxin B resistance

Shigella spp. are the causative agent of bacillary dysentery, a major cause of food-borne morbidity and mortality worldwide. These organisms are recently evolved, polyphyletic pathovar of E. coli, and since their divergence they have undergone multiple cases of gene gain and gene loss and understanding how gene inactivation events alter bacterial behaviour represents an important objective to be better able to understand how virulence and other phenotypes are affected. Here, we identify a frameshift mutation in the pmrD gene of S. flexneri that although it would be predicted to make a functional, full-length protein, no such production occurs, likely due to the non-optimal spacing between the translational initiation site and the Shine-Dalgarno sequence. We show that this loss severs the normal connection between the PhoPQ two-component regulatory system and the PmrAB two-component regulatory system, abrogating low Mg2+ mediated cationic antimicrobial peptide and polymyxin B resistance, while maintaining normal PmrAB-mediated polymyxin B resistance. In contrast, S. sonnei maintains a functional PmrD protein and canonical signaling through this regulatory network. This species specific gene loss suggests that S. flexneri and S. sonnei have evolved different regulatory responses to changing environmental conditions.

microbiology↗

bioRxiv2021

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease

The COVID-19 pandemic has led to an urgent need for the identification of new antiviral drug therapies that can be rapidly deployed to treat patients with this disease. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of COVID-19. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cellbased experimental assessment reveals several clinically-relevant repurposing drug candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

cell biology↗

bioRxiv2020

Conserved regulation of omptin proteases by the PhoPQ two-component regulatory system in Enterobacteriaceae.

Bacteria that colonize eukaryotic surfaces interact with numerous host-produced molecules that have antimicrobial activity. Bacteria have evolved numerous strategies to both detect and resist these molecules, and in gram-negative bacteria these include alterations of the cell surface lipopolysaccharide structure and/or charge and the production of proteases that can degrade these antimicrobial molecules. Many of the lipopolysaccharide alterations found in enteric bacteria are controlled by the PhoPQ and PmrAB two-component regulatory systems. Here, we show that omptin family proteases from Escherichia coli and Citrobacter rodentium are induced by growth in low Mg2+. We further show that deletion of PhoP eliminates omptin protease activity, transcriptional regulation and protein levels. We identify conserved PhoP-binding sites in the promoters of the E. coli omptin genes, ompT, ompP and arlC as well as in croP of Citrobacter rodentium and show that mutation of the putative PhoP-binding site in the ompT promoter abrogates PhoP-dependent expression. Finally, we show that despite the conserved PhoP-dependent regulation, each of the E. coli omptin proteins has differential activity toward a particular substrate, suggesting that each omptin may contribute to resistance to a particular repertoire of host-defense peptides, depending on the particular environment in which each evolved.

microbiology↗