Structure-guided discovery of anti-CRISPR and anti-phage defense proteins.

Bacteria use a variety of defense systems to protect themselves from phage infection. In turn, phages have evolved diverse counter-defense measures to overcome host defenses. Here, we use protein structural similarity and gene co-occurrence analyses to screen >66 million viral protein sequences and >330,000 metagenome-assembled genomes for the identification of anti-phage and counter-defense systems. We predict structures for ~300,000 proteins and perform large-scale, pairwise comparison to known anti-CRISPR (Acr) and anti-phage proteins to identify structural homologs that otherwise may not be uncovered using primary sequence search. This way, we identify a Bacteroidota phage Acr protein that inhibits Cas12a, and an Akkermansia muciniphila anti-phage defense protein, termed BxaP. Gene bxaP is found in loci encoding Bacteriophage Exclusion (BREX) and restriction-modification defense systems, but confers immunity independently. Our work highlights the advantage of combining protein structural features and gene co-localization information in studying host-phage interactions.

Skin Microbiome Variation with Cancer Progression in Human Cutaneous Squamous Cell Carcinoma.

The skin microbiome plays a critical role in skin homeostasis and disorders. UVR is the major cause of nonmelanoma skin cancer, but other risk factors, including immune suppression, chronic inflammation, and antibiotic usage, suggest the microbiome as an additional, unexplored risk factor and potential disease biomarker. The overarching goal was to study the skin microbiome in squamous cell carcinoma (SCC) and premalignant actinic keratosis compared with that in healthy skin to identify skin cancer‒associated changes in the skin microbiome. We performed a high-resolution analysis of shotgun metagenomes of actinic keratosis and SCC in healthy skin, revealing the microbial community shifts specific to actinic keratosis and SCC. Most prominently, the relative abundance of pathobiont Staphylococcus aureus was increased at the expense of commensal Cutibacterium acnes in SCC compared with that in healthy skin, and enrichment of functional pathways in SCC reflected this shift. Notably, C. acnes associated with lesional versus healthy skin differed at the strain level, suggesting the specific functional changes associated with its depletion in SCC. Our study revealed a transitional microbial dysbiosis from healthy skin to actinic keratosis to SCC, supporting further investigation of the skin microbiome for use as a biomarker and providing hypotheses for studies investigating how these microbes might influence skin cancer progression.

Metagenomic growth rate inferences of strains in situ.

We developed a method for strain-level metagenomic estimation of growth rate (SMEG) for inferring growth rates of bacterial subspecies, or strains, from complex metagenomic samples. We applied our method, which is based on both reference strains and de novo approaches, to different gut metagenomic datasets, accurately identifying an outbreak-associated Escherichia coli strain and a previously unidentified association of an Akkermansia muciniphila strain in cancer immunotherapy responders. SMEG resolves strain-specific growth rates from mixtures of commensal or pathogenic strains to provide new insights into microbial interactions and disease associations at the strain level. SMEG is available for download at https://github.com/ohlab/SMEG.

High throughput in situ metagenomic measurement of bacterial replication at ultra-low sequencing coverage.

We developed Growth Rate InDex (GRiD) for estimating in situ growth rates of ultra-low coverage (>0.2×) and de novo-assembled metagenomes. Applying GRiD to human and environmental metagenomic datasets to demonstrate its versatility, we uncovered new associations with previously uncharacterized bacteria whose growth rates were associated with several disease characteristics or environmental interactions. In addition, with GRiD-MG (metagenomic), a high-throughput implementation of GRiD, we estimated growth dynamics of 1756 bacteria species from a healthy skin metagenomic dataset and identified a new Staphylococcus-Corynebacterium antagonism likely mediated by antimicrobial production in the skin. GRiD-MG significantly increases the ability to extract growth rate inferences from complex metagenomic data with minimal input from the user.

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli.

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.

A Complete Pathway Model for Lipid A Biosynthesis in Escherichia coli.

Lipid A is a highly conserved component of lipopolysaccharide (LPS), itself a major component of the outer membrane of Gram-negative bacteria. Lipid A is essential to cells and elicits a strong immune response from humans and other animals. We developed a quantitative model of the nine enzyme-catalyzed steps of Escherichia coli lipid A biosynthesis, drawing parameters from the experimental literature. This model accounts for biosynthesis regulation, which occurs through regulated degradation of the LpxC and WaaA (also called KdtA) enzymes. The LpxC degradation signal appears to arise from the lipid A disaccharide concentration, which we deduced from prior results, model results, and new LpxK overexpression results. The model agrees reasonably well with many experimental findings, including the lipid A production rate, the behaviors of mutants with defective LpxA enzymes, correlations between LpxC half-lives and cell generation times, and the effects of LpxK overexpression on LpxC concentrations. Its predictions also differ from some experimental results, which suggest modifications to the current understanding of the lipid A pathway, such as the possibility that LpxD can replace LpxA and that there may be metabolic channeling between LpxH and LpxB. The model shows that WaaA regulation may serve to regulate the lipid A production rate when the 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) concentration is low and/or to control the number of KDO residues that get attached to lipid A. Computation of flux control coefficients showed that LpxC is the rate-limiting enzyme if pathway regulation is ignored, but that LpxK is the rate-limiting enzyme if pathway regulation is present, as it is in real cells. Control also shifts to other enzymes if the pathway substrate concentrations are not in excess. Based on these results, we suggest that LpxK may be a much better drug target than LpxC, which has been pursued most often.

A model for the proteolytic regulation of LpxC in the lipopolysaccharide pathway of Escherichia coli.

Lipopolysaccharide (LPS) is an essential structural component found in Gram-negative bacteria. The molecule is comprised of a highly conserved lipid A and a variable outer core consisting of various sugars. LPS plays important roles in membrane stability in the bacterial cell and is also a potent activator of the human immune system. Despite its obvious importance, little is understood regarding the regulation of the individual enzymes involved or the pathway as a whole. LpxA and LpxC catalyze the first two steps in the LPS pathway. The reaction catalyzed by LpxA possesses a highly unfavourable equilibrium constant with no evidence of coupling to an energetically favourable reaction. In our model the presence of the second enzyme LpxC was sufficient to abate this unfavourable reaction and confirming previous studies suggesting that this reaction is the first committed step in LPS synthesis. It is believed that the protease FtsH regulates LpxC activity via cleavage. It is also suspected that the activity of FtsH is regulated by a metabolite produced by the LPS pathway; however, it is not known which one. In order to investigate these mechanisms, we obtained kinetic parameters from literature and developed estimates for other simulation parameters. Our simulations suggest that under modest increases in LpxC activity, FtsH is able to regulate the rate of product formation. However, under extreme increases in LpxC activities such as over-expression or asymmetrical cell division then FtsH activation may not be sufficient to regulate this first stage of synthesis.