Plasma Membrane-Cell Wall Feedback in Bacteria.

Most bacteria have cell wall peptidoglycan surrounding their plasma membranes. The essential cell wall provides a scaffold for the envelope, protection against turgor pressure and is a proven drug target. Synthesis of the cell wall involves reactions that span cytoplasmic and periplasmic compartments. Bacteria carry out the last steps of cell wall synthesis along their plasma membrane. The plasma membrane in bacteria is heterogeneous and contains membrane compartments. Here, I outline findings that highlight the emerging notion that plasma membrane compartments and the cell wall peptidoglycan are functionally intertwined. I start by providing models of cell wall synthesis compartmentalization within the plasma membrane in mycobacteria, Escherichia coli, and Bacillus subtilis. Then, I revisit literature that supports a role for the plasma membrane and its lipids in modulating enzymatic reactions that synthesize cell wall precursors. I also elaborate on what is known about bacterial lateral organization of the plasma membrane and the mechanisms by which organization is established and maintained. Finally, I discuss the implications of cell wall partitioning in bacteria and highlight how targeting plasma membrane compartmentalization serves as a way to disrupt cell wall synthesis in diverse species.

Cell Wall Damage Reveals Spatial Flexibility in Peptidoglycan Synthesis and a Nonredundant Role for RodA in Mycobacteria.

Cell wall peptidoglycan is a heteropolymeric mesh that protects the bacterium from internal turgor and external insults. In many rod-shaped bacteria, peptidoglycan synthesis for normal growth is achieved by two distinct pathways: the Rod complex, comprised of MreB, RodA, and a cognate class B penicillin-binding protein (PBP), and the class A PBPs (aPBPs). In contrast to laterally growing bacteria, pole-growing mycobacteria do not encode an MreB homolog and do not require SEDS protein RodA for in vitro growth. However, RodA contributes to the survival of Mycobacterium tuberculosis in some infection models, suggesting that the protein could have a stress-dependent role in maintaining cell wall integrity. Under basal conditions, we find here that the subcellular distribution of RodA largely overlaps that of the aPBP PonA1 and that both RodA and the aPBPs promote polar peptidoglycan assembly. Upon cell wall damage, RodA fortifies Mycobacterium smegmatis against lysis and, unlike aPBPs, contributes to a shift in peptidoglycan assembly from the poles to the sidewall. Neither RodA nor PonA1 relocalize; instead, the redistribution of nascent cell wall parallels that of peptidoglycan precursor synthase MurG. Our results support a model in which mycobacteria balance polar growth and cell-wide repair via spatial flexibility in precursor synthesis and extracellular insertion. IMPORTANCE Peptidoglycan synthesis is a highly successful target for antibiotics. The pathway has been extensively studied in model organisms under laboratory-optimized conditions. In natural environments, bacteria are frequently under attack. Moreover, the vast majority of bacterial species are unlikely to fit a single paradigm of cell wall assembly because of differences in growth mode and/or envelope structure. Studying cell wall synthesis under nonoptimal conditions and in nonstandard species may improve our understanding of pathway function and suggest new inhibition strategies. Mycobacterium smegmatis, a relative of several notorious human and animal pathogens, has an unusual polar growth mode and multilayered envelope. In this work, we challenged M. smegmatis with cell wall-damaging enzymes to characterize the roles of cell wall-building enzymes when the bacterium is under attack.

The Cantaloupe Farm Environment Has a Diverse Genetic Pool of Antibiotic-Resistance and Virulence Genes.

Cantaloupes contaminated with pathogens have led to many high-profile outbreaks and illnesses. Since bacterial virulence genes (VGs) can act in tandem with antibiotic-resistance and mobile genetic elements, there is a need to evaluate these gene reservoirs in fresh produce, such as cantaloupes. The goal of this study was to assess the distribution of antibiotic-resistance, virulence, and mobile genetic elements genes (MGEGs) in cantaloupe farm environments. A total of 200 samples from cantaloupe melons (n = 99), farm workers' hands (n = 66), and production water (n = 35) were collected in México. Each sample was assayed for the presence of 14 antibiotic-resistance genes, 15 VGs, and 5 MGEGs by polymerase chain reaction. Our results indicated that tetracycline (tetA and tetB) (18% of cantaloupe, 45% of hand samples) and sulfonamide (sul1) (30% of cantaloupe, 71% of hand samples) resistance genes were frequently detected. The colistin resistance gene (mcr1) was detected in 10% of cantaloupe and 23% of farm workers' hands. Among VGs, Salmonella genes invA and spiA were the most abundant. There was a significantly higher likelihood of detecting antibiotic-resistance, virulence, and MGEGs on hands compared with water samples. These results demonstrate a diverse pool of antibiotic-resistance and VGs in cantaloupe production.

Natural plant products inhibits growth and alters the swarming motility, biofilm formation, and expression of virulence genes in enteroaggregative and enterohemorrhagic Escherichia coli.

The purpose of this study was to determine the effects of plant products on the growth, swarming motility, biofilm formation and virulence gene expression in enterohemorrhagic Escherichia coli O157:H7 and enteroaggregative E. coli strain 042 and a strain of O104:H4 serotype. Extracts of Lippia graveolens and Haematoxylon brassiletto, and carvacrol, brazilin were tested by an antimicrobial microdilution method using citral and rifaximin as controls. All products showed bactericidal activity with minimal bactericidal concentrations ranging from 0.08 to 8.1 mg/ml. Swarming motility was determined in soft LB agar. Most compounds reduced swarming motility by 7%-100%; except carvacrol which promoted motility in two strains. Biofilm formation studies were done in microtiter plates. Rifaximin inhibited growth and reduced biofilm formation, but various concentrations of other compounds actually induced biofilm formation. Real time PCR showed that most compounds decreased stx2 expression. The expression of pic and rpoS in E. coli 042 were suppressed but in E. coli O104:H4 they varied depending on compounds. In conclusion, these extracts affect E. coli growth, swarming motility and virulence gene expression. Although these compounds were bactericidal for pathogenic E. coli, sublethal concentrations had varied effects on phenotypic and genotypic traits, and some increased virulence gene expression.

Natural and synthetic antimicrobials reduce adherence of enteroaggregative and enterohemorrhagic Escherichia coli to epithelial cells.

Adherence of bacteria to the human intestinal mucosa can facilitate their internalization and the development of pathological processes. Escherichia coli O104:H4 is considered a hybrid strain (enteroaggregative hemorrhagic E. coli [EAHEC]), sharing virulence factors found in enterohemorrhagic (EHEC), and enteroaggregative (EAEC) E. coli pathotypes. The objective of this study was to analyze the effects of natural and synthetic antimicrobials (carvacrol, oregano extract, brazilin, palo de Brasil extract, and rifaximin) on the adherence of EHEC O157:H7, EAEC 042, and EAHEC O104:H4 to HEp-2 cells and to assess the expression of various genes involved in this process. Two concentrations of each antimicrobial that did not affect (p≤0.05) bacterial viability or damage the bacterial membrane integrity were used. Assays were conducted to determine whether the antimicrobials alter adhesion by affecting bacteria and/or alter adhesion by affecting the HEp-2 cells, whether the antimicrobials could detach bacteria previously adhered to HEp-2 cells, and whether the antimicrobials could modify the adherence ability exhibited by the bacteria for several cycles of adhesion assays. Giemsa stain and qPCR were used to assess the adhesion pattern and gene expression, respectively. The results showed that the antimicrobials affected the adherence abilities of the bacteria, with carvacrol, oregano extract, and rifaximin reducing up to 65% (p≤0.05) of E. coli adhered to HEp-2 cells. Carvacrol (10 mg/ml) was the most active compound against EHAEC O104:H4, even altering its aggregative adhesion pattern. There were changes in the expression of adhesion-related genes (aggR, pic, aap, aggA, and eae) in the bacteria and oxidative stress-related genes (SOD1, SOD2, CAT, and GPx) in the HEp-2 cells. In general, we demonstrated that carvacrol, oregano extract, and rifaximin at sub-minimal bactericidal concentrations interfere with target sites in E. coli, reducing the adhesion efficiency.

Membrane-partitioned cell wall synthesis in mycobacteria.

Many antibiotics target the assembly of cell wall peptidoglycan, an essential, heteropolymeric mesh that encases most bacteria. In rod-shaped bacteria, cell wall elongation is spatially precise yet relies on limited pools of lipid-linked precursors that generate and are attracted to membrane disorder. By tracking enzymes, substrates, and products of peptidoglycan biosynthesis in Mycobacterium smegmatis, we show that precursors are made in plasma membrane domains that are laterally and biochemically distinct from sites of cell wall assembly. Membrane partitioning likely contributes to robust, orderly peptidoglycan synthesis, suggesting that these domains help template peptidoglycan synthesis. The cell wall-organizing protein DivIVA and the cell wall itself promote domain homeostasis. These data support a model in which the peptidoglycan polymer feeds back on its membrane template to maintain an environment conducive to directional synthesis. Our findings are applicable to rod-shaped bacteria that are phylogenetically distant from M. smegmatis, indicating that horizontal compartmentalization of precursors may be a general feature of bacillary cell wall biogenesis.

Supramolecular antibiotics: a strategy for conversion of broad-spectrum to narrow-spectrum antibiotics for Staphylococcus aureus.

The propensity of broad-spectrum antibiotics to indiscriminately kill both pathogenic and beneficial bacteria has a profound impact on the spread of resistance across multiple bacterial species. Alternative approaches that narrow antibacterial specificity towards desired pathogenic bacterial population are of great interest. Here, we report an enzyme-responsive antibiotic-loaded nanoassembly strategy for narrow delivery of otherwise broad-spectrum antibiotics. We specifically target Staphylococcus aureus (S. aureus), an important blood pathogen that secretes PC1 ß-lactamases. Our nanoassemblies selectively eradicate S. aureus grown in vitro with other bacteria, highlighting its potential capability in targeting the desired pathogenic bacterial population.

Phylogroups, pathotypes, biofilm formation and antimicrobial resistance of Escherichia coli isolates in farms and packing facilities of tomato, jalapeño pepper and cantaloupe from Northern Mexico.

The most commonly used indicator of fecal contamination in fresh produce production and packing is Escherichia coli. In depth analysis of the prevalence and characteristics of naturally occurring E. coli strains in these environments is important because it can (1) serve as an indicator of sources of fecal contamination; and (2) provide information on strain pathogenicity, persistence, and other defining characteristics such as multidrug resistance. In this study, we analyzed 341 E. coli strains isolated from the jalapeño pepper, tomato and cantaloupe farm environments, in Northeast Mexico. Strains were isolated from produce, farmworkers' hands, soil and water. Pathotypes, genotypes, biofilm formation and antibiotic resistance were characterized. Phylogenetic subgroups and identification of diarrheagenic E. coli were determined by PCR; biofilm formation was quantified using a plate-based colorimetric method. Antibiotic resistance was analyzed by the Kirby Bauer diffusion disc method. Most isolates (N = 293, 86%) belonged to phylogenetic group A. Only four isolates (1.2%) were diarrheagenic: EPEC (N = 3) and ETEC (N = 1). Antibiotic resistance to tetracycline (23.2%) and ampicillin (19.9%) was high, and only 3.5% of the strains presented resistance to >5 antibiotics. Biofilms were produced by most strains (76%), among which 34.4% were categorized as high producers. The presence of antibiotic resistant E. coli strains that may contain gene markers for pathogenicity and which can form biofilms suggests potential health risks for consumers.

Spatial control of cell envelope biosynthesis in mycobacteria.

The mycobacterial cell envelope is a complex multilayered structure that provides the strength to the rod-shaped cell and creates the permeability barrier against antibiotics and host immune attack. In this review, we will discuss the spatial coordination of cell envelope biosynthesis and how plasma membrane compartmentalization plays a role in this process. The spatial organization of cell envelope biosynthetic enzymes as well as other membrane-associated proteins is crucial for cellular processes such as polar growth and midcell septum formation. We will highlight metabolic enzymes involved in the localized biosynthesis of envelope components such as peptidoglycan, arabinogalactan and outer/inner membrane lipids. The known and potential roles of cytoskeletal and coiled coil proteins in driving subcellular protein localization will also be summarized. Finally, we provide a comprehensive overview of known lateral heterogeneities in mycobacterial plasma membrane, with a particular focus on the intracellular membrane domain, recently revealed by biochemical fractionation and fluorescence microscopy. We consider how this dynamic and multifunctional membrane microdomain contributes to the subcellular localization of membrane proteins and spatially restricted cell envelope biosynthesis in mycobacteria.

Peptidoglycan precursor synthesis along the sidewall of pole-growing mycobacteria.

Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.