Biology subjects

Patel, D. H.

Publications and source records attributed to Patel, D. H..

4 recordsLinked to original sources

bioRxiv2024

Cryo-EM Structure of Salmonella typhimurium ArnC; the Key Enzyme in Lipid-A Modification Conferring Polymyxin Resistance

1Polymyxins are last-resort antimicrobial peptides administered clinically against multi-drug resistant bacteria, including Gram-negative ESKAPE pathogens. However, an increasing number of pathogens employ a defense strategy involving a relay of enzymes encoded by the pmrE(ugd) loci and the arnBCDTEF operon. As a result, an Ara-4N headgroup is added to the lipid-A component of outer membrane (OM) lipopolysaccharides (LPS) rendering polymyxins ineffective. Here, we report the cryo-EM structures of glycosyltransferase ArnC from Salmonella typhimurium resolved in both apo and UDP-bound forms at resolutions 2.75 [A] and 3.8 [A], respectively. The structure of the ArnC protomer comprises of three distinct regions: an N-terminal glycosyltransferase domain, transmembrane region, and the interface helices (IHs). ArnC forms a stable tetramer with C2 symmetry through interactions in the C-terminal region, which is expected to protrude into the cytosol, where the {beta}8 strand inserts into the adjacent protomer. ArnC protomers have two distinct types of interfaces involving multiple hydrogen bonds and salt bridges. The binding of UDP induces conformational changes that stabilizes structurally labile A-loop, spanning residues 201 to 213, and part of the putative catalytic pocket formed by IH1 and IH2. The comparative analysis of ArnC structures with homologs GtrB and DPMS suggests the key residues involved in ArnC catalytic activity.

biochemistry↗

bioRxiv2024

Global atlas of predicted functional domains in Legionella pneumophila Dot/Icm translocated effectors

Legionella pneumophila utilizes the Dot/Icm type IVB secretion system to deliver hundreds of effector proteins inside eukaryotic cells to ensure intracellular replication. Our understanding of the molecular functions of this largest pathogenic arsenal known to the bacterial world remains incomplete. By leveraging advancements in 3D protein structure prediction, we provide a comprehensive structural analysis of 368 L. pneumophila effectors, representing a global atlas of predicted functional domains summarized in a database (https://pathogens3d.org/legionella-pneumophila). Our analysis identified 157 types of diverse functional domains in 287 effectors, including 159 effectors with no prior functional annotations. Furthermore, we identified 35 unique domains in 30 effector models that have no similarity with experimentally structurally characterized proteins, thus, hinting at novel functionalities. Using this analysis, we demonstrate the activity of thirteen domains, including three unique folds, predicted in L. pneumophila effectors to cause growth defects in the Saccharomyces cerevisiae model system. This illustrates an emerging strategy of exploring synergies between predictions and targeted experimental approaches in elucidating novel effector activities involved in infection.

microbiology↗

bioRxiv2023

Structural characterization of the Sel1-like repeat protein LceB from Legionella pneumophila

Legionella are freshwater Gram-negative bacteria that in their normal environment infect protozoa. However, this adaptation also allows Legionella to infect human alveolar macrophages and cause pneumonia. Central to Legionella pathogenesis are more than 330 secreted effectors, of which there are 9 core effectors that are conserved in all pathogenic species. Despite their importance, the biochemical function of several core effectors remains unclear. To address this, we have taken a structural approach to characterize the core effector of unknown function LceB, or Lpg1356, from Legionella pneumophila. Here we solve an X-ray crystal structure of LceB using an AlphaFold model for molecular replacement. The experimental structure shows that LceB adopts a Sel1-like repeat fold as predicted. However, the crystal structure captured multiple conformations of LceB all of which differed from the AlphaFold model. Comparison of the predicted model and the experimental models suggests that LceB is highly flexible in solution. Additionally, molecular analysis of LceB using its close structural homologues reveals sequence and structural motifs of known biochemical function. Specifically, LceB harbors a repeated KAAEQG motif that both stabilizes the Sel1-like repeat fold and is known to participate in protein-protein interactions with eukaryotic host proteins. We also observe that LceB forms several higher-order oligomers in solution. Overall, our results have revealed that LceB has conformational flexibility, self-associates, and contains a molecular surface for binding a target host-cell protein. Additionally, our data provides structural insights into the Sel1-like repeat family of proteins that remain poorly studied.

biochemistry↗

bioRxiv2021

Blocking mitophagy does not improve fuel ethanol production in Saccharomyces cerevisiae

Ethanol fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Shiroma and co-workers (2014) showed that blocking mitophagy by deletion of the mitophagy specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no differences in CO2 production, final ethanol titres or cell viability were observed after five rounds of ethanol fermentation, cell recycling and acid treatment, as commonly performed in sugarcane biorefineries. To test if S. cerevisiaes strain background influences this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial) and BY (laboratory), with and without a functional ATG32 gene, under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability and ethanol titres, among the three strains, we could not observe any improvement in fermentation performance related to the blocking of mitophagy. We conclude with caution that results obtained with Ginjo sake yeast is an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation. ImportanceBioethanol is the largest (per volume) ever biobased bulk chemical produced globally. The fermentation process is very well established, and industries regularly attain nearly 85% of maximum theoretical yields. However, because of the volume of fuel produced, even a small improvement will have huge economic benefits. To this end, besides already implemented process improvements, various free energy conservation strategies have been successfully exploited at least in laboratory strains to increase ethanol yields and decrease by-product formation. Cellular housekeeping processes have been an almost unexplored territory in strain improvement. Shiroma and co-workers previously reported that blocking mitophagy by deletion of the mitophagy receptor gene ATG32 in Saccharomyces cerevisiae led to a 2.12% increase in final ethanol titres during Japanese sake fermentation. We found in two commercially used bioethanol strains (PE-2 and Ethanol Red) that ATG32 deficiency does not lead to an improvement in cell viability or ethanol levels during fermentation with molasses or in a synthetic complete medium. More research is required to ascertain the role of autophagic processes during fermentation conditions.

microbiology↗