Biology subjects

Knodler, L. A.

Publications and source records attributed to Knodler, L. A..

4 recordsLinked to original sources

bioRxiv2025

SLC11A2 affects nutritional immunity in the gastrointestinal epithelium

There is a constant tug-of-war for transition metals at the pathogen-host interface. A goal of the vertebrate host is to modulate the availability of metals to pathogens, in a process known as nutritional immunity, but pathogens have evolved numerous countermeasures to regulate intracellular trace metal levels. The bioavailability of trace metals therefore shapes the outcome of disease. In the human body, epithelial cells lining the intestine are a major site of metal absorption. Intestinal epithelial cells (IECs) are also a target for invading enteric pathogens but the contribution of epithelium-intrinsic factors towards nutritional immunity has been understudied. Using Salmonella enterica serovar Typhimurium (STm) harboring metal-responsive fluorescent reporters in a bovine ligated intestinal loop infection model, we mapped the spatiotemporal nature of metal competition during enteric salmonellosis. We show that STm experiences a temporal, cell-specific restriction of iron, manganese, and zinc in the intestinal mucosa during the early stages of infection. We have further studied the contribution of the broad specificity metal cation transporter, SLC11A2, in IECs to nutritional immunity against STm. Knockout of SLC11A2 in IECs leads to enhanced replication of STm, indicating a protective role for this transporter. Using fluorescence-based biosensors and bacterial gene deletion mutants, we pinpoint manganese and iron restriction as the mechanism by which SLC11A2 limits bacterial proliferation. We conclude that SLC11A2-mediated sequestration of metals is an intrinsic defense mechanism of the intestinal epithelium against enteric bacteria. Significance statementThere is limited source of trace metals in the gut that invading pathogens and the infected host must compete for. Using Salmonella enterica as a model enteric pathogen, along with fluorescent reporters that respond to metal ion availability, we traced the sites of metal ion limitation in the intestinal mucosa to intestinal epithelial cells (IECs) and phagocytes in the underlying lamina propria. We further show that SLC11A2 (NRAMP2), which is the sole SLC11 family member expressed in IECs and localizes to the apical membrane and endosomal network, limits the intracellular proliferation of Salmonella enterica by withholding iron and manganese. Therefore SLC11A2-mediated nutritional immunity is an IEC-intrinsic defense mechanism that protects against microbial pathogens.

microbiology↗

bioRxiv2024

Pathogenic diversification of the gut commensal Providencia alcalifaciens via acquisition of a second type III secretion system

Providencia alcalifaciens is a Gram-negative bacterium found in a wide variety of water and land environments and organisms. It has been isolated as part of the gut microbiome of animals and insects, as well as from stool samples of patients with diarrhea. Specific P. alcalifaciens strains encode gene homologs of virulence factors found in other pathogenic members of the same Enterobacterales order, such as Salmonella enterica serovar Typhimurium and Shigella flexneri. Whether these genes are also pathogenic determinants in P. alcalifaciens is not known. Here we have used P. alcalifaciens 205/92, a clinical isolate, with in vitro and in vivo infection models to investigate P. alcalifaciens-host interactions at the cellular level. Our particular focus was the role of two type III secretion systems (T3SS) belonging to the Inv-Mxi/Spa family. T3SS1b is widespread in Providencia spp. and encoded on the chromosome. T3SS1a is encoded on a large plasmid that is present in a subset of P. alcalifaciens strains, which are primarily isolates from diarrheal patients. Using a combination of electron and fluorescence microscopy and gentamicin protection assays we show that P. alcalifaciens 205/92 is internalized into eukaryotic cells, rapidly lyses its internalization vacuole and proliferates in the cytosol. This triggers caspase-4 dependent inflammasome responses in gut epithelial cells. The requirement for the T3SS1a in entry, vacuole lysis and cytosolic proliferation is host-cell type specific, playing a more prominent role in human intestinal epithelial cells as compared to macrophages. In a bovine ligated intestinal loop model, P. alcalifaciens colonizes the intestinal mucosa, inducing mild epithelial damage with negligible fluid accumulation. No overt role for T3SS1a or T3SS1b was seen in the calf infection model. However, T3SS1b was required for the rapid killing of Drosophila melanogaster. We propose that the acquisition of two T3SS by horizontal gene transfer has allowed P. alcalifaciens to diversify its host range, from a highly virulent pathogen of insects to an opportunistic gastrointestinal pathogen of animals.

microbiology↗

bioRxiv2022

Non-redundant dimethyl sulfoxide reductases influence Salmonella enterica serotype Typhimurium anaerobic growth and virulence

Facultative anaerobic enteric pathogens can utilize a diverse array of alternate electron acceptors to support anaerobic metabolism and thrive in the hypoxic conditions within the mammalian gut. Dimethyl sulfoxide (DMSO) is produced by methionine catabolism and can act as an alternate electron acceptor to support anaerobic respiration. The DMSO reductase complex consists of three subunits, DmsA, DmsB, and DmsC, and allows bacteria to grow anaerobically with DMSO as an electron acceptor. The genomes of non-typhoidal S. enterica encode three putative dmsABC operons, but the impact of the apparent genetic redundancy in DMSO reduction on fitness of non-typhoidal S. enterica during infection remains unknown. We hypothesized that DMSO reduction would be needed for S. enterica serotype Typhimurium (S. Typhimurium) to colonize the mammalian gut. We demonstrate a S. Typhimurium mutant with loss of function in all three putative DMSO reductases ({Delta}dmsA3) poorly colonizes the mammalian intestine when the microbiota are intact and when inflammation is absent. DMSO reduction enhances anaerobic growth through non-redundant contributions of two of the DMSO reductases. Furthermore, DMSO reduction influences the expression of the type-3 secretion systems needed for virulence. Collectively, our data demonstrate that the DMSO reductases of S. Typhimurium are functionally non-redundant and suggest DMSO is a physiologically relevant electron acceptor that supports S. enterica fitness in the gut.

microbiology↗

bioRxiv2020

Global mapping of Salmonella enterica-host protein-protein interactions during infection

Intracellular bacterial pathogens inject effector proteins into host cells to hijack diverse cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effector proteins, and quantified PPIs in macrophages and epithelial cells by Affinity-Purification Quantitative Mass-Spectrometry. Thereby, we identified 25 previously described and 421 novel effector-host PPIs. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. Using reciprocal co-immunoprecipitations, we validated 13 out of 22 new PPIs. We then used this host-pathogen physical interactome resource to demonstrate that SseJ and SseL collaborate in redirecting cholesterol to the Salmonella Containing Vacuole (SCV) via NPC1, PipB directly recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by directly phosphorylating formin-like proteins.

microbiology↗