The Cyclopropane Fatty Acid Synthase Mediates Antibiotic Resistance and Gastric Colonization of Helicobacter pylori

ABSTRACT Cyclopropane fatty acids (CFAs) are synthetized by the addition of a methylene group from S-adenosyl-l-methionine across the carbon-carbon double bonds of unsaturated fatty acid chains of membrane phospholipids. This fatty acid cyclopropanation, catalyzed by the CFA synthase (CfaS) enzyme, occurs in many bacteria, including the human pathogen Helicobacter pylori. Although the cyclopropane modification was reported to play a key role in the adaptation in response to environmental stress, its role in H. pylori remains unknown. In this study, we showed that H. pylori HP0416 encodes a functional CfaS. The enzyme was demonstrated to be required for acid resistance, antibiotic resistance, intracellular survival and mouse gastric colonization, and cell membrane integrity. Moreover, the tool compound dioctylamine, which acts as a substrate mimic, directly inhibits the CfaS function of H. pylori, resulting into sensitivity to acid stress, increased antibiotic susceptibility, and attenuated abilities to avoid macrophage killing and to colonize mouse stomachs. These results validate CfaS as a promising antibiotic target and provide new potentials for this recognized target in future anti-H. pylori drug discovery efforts. IMPORTANCE The increasing prevalence of multidrug-resistant Helicobacter pylori strains has created an urgent need for alternative therapeutic regimens that complement the current antibiotic treatment strategies for H. pylori eradication; however, this is greatly hampered due to a lack of “druggable” targets. Although the CFAs are present in H. pylori cytoplasmic membranes at high levels, their physiological role has not been established. In this report, deletion of the CFA synthase CfaS was shown to attenuate acid and drug resistance, immune escape, and gastric colonization of H. pylori. These findings were validated by inhibition of the CfaS activity with the tool compound dioctylamine. These studies identify this enzyme as an attractive target for further drug discovery efforts against H. pylori.

Gastric Dilatation Associated with Gastric Colonization with Sarcina-Like Bacteria in a Cat with Chronic Enteritis

ABSTRACT An 11 yr old spayed female domestic longhair cat was presented for an acute onset of vomiting. Abdominal radiographs and ultrasound revealed severe gastric dilatation (GD) without evidence of gastric outflow obstruction. On esophagogastroduodenoscopy, the duodenal mucosa was mildly erythematous, and a moderate, diffuse, chronic enteritis was found by histological examination of duodenal biopsies. Large numbers of Sarcina-like bacteria without associated inflammation were present in gastric mucosal biopsies. To the authors’ knowledge, this is the first report of GD associated with colonization by Sarcina-like bacteria in a cat. Gastric colonization by Sarcina-like bacteria should be suspected when cats are presented with acute onset of GD and vomiting.

A Burkholderia pseudomallei Colony Variant Necessary for Gastric Colonization

ABSTRACT Diverse colony morphologies are a hallmark ofBurkholderia pseudomalleirecovered from infected patients. We observed that stresses that inhibit aerobic respiration shifted populations of B. pseudomallei from the canonical white colony morphotype toward two distinct, reversible, yet relatively stable yellow colony variants (YA and YB). As accumulating evidence supports the importance of B. pseudomallei enteric infection and gastric colonization, we tested the response of yellow variants to hypoxia, acidity, and stomach colonization. Yellow variants exhibited a competitive advantage under hypoxic and acidic conditions and alkalized culture media. The YB variant, although highly attenuated in acute virulence, was the only form capable of colonization and persistence in the murine stomach. The accumulation of extracellular DNA (eDNA) was a characteristic of YB as observed by 4′,6-diamidino-2-phenylindole (DAPI) staining of gastric tissues, as well as in anin vitrostomach model where large amounts of eDNA were produced without cell lysis. Transposon mutagenesis identified a transcriptional regulator (BPSL1887, designated YelR) that when overexpressed produced the yellow phenotype. Deletion ofyelRblocked a shift from white to the yellow forms. These data demonstrate that YB is a unique B. pseudomallei pathovariant controlled by YelR that is specifically adapted to the harsh gastric environment and necessary for persistent stomach colonization.IMPORTANCE Seemingly uniform populations of bacteria often contain subpopulations that are genetically identical but display unique characteristics which offer advantages when the population is faced with infrequent but predictable stresses. The pathogenBurkholderia pseudomalleiis capable of forming several reversible colony types, and it interconverted between one white type and two yellow types under certain environmental stresses. The two yellow forms exhibited distinct advantages in low-oxygen and acidic environments. One yellow colony variant was the only form capable of chronic stomach colonization. Areas of gastric infection were marked by bacteria encased in a DNA matrix, and the yellow forms were able to produce large amounts of extracellular DNAin vitro. We also identified the regulator in control of yellow colony variant formation. These findings demonstrate a role in infection for colony variation and provide a mechanism for chronic stomach colonization—a frequently overlooked niche in melioidosis.