Peptidoglycan is an essential structural component of the cell wall in the majority of bacteria, but the obligate intracellular human pathogen Chlamydia trachomatis was thought to be one of the few exceptions; here a click chemistry approach is used to label peptidoglycan in replicating C. trachomatis with novel d-amino acid dipeptide probes. The sugar amino acid polymer peptidoglycan is an essential cell-wall component in most free-living bacteria. The Chlamydiales, Gram-negative parasites including the human pathogen Chlamydia trachomatis, were thought to be a rare exception: they encode genes for peptidoglycan biosynthesis and are susceptible to β-lactam antibiotics, yet attempts to detect chlamydial peptidoglycans had failed. Now this paradox, known as the 'chlamydial anomaly', has been resolved. This study, using a novel click chemistry technique to label peptidoglycans with D-amino acid dipeptide probes, demonstrates the presence of peptidoglycans in replicating C. trachomatis. Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia harbour genes for PG biosynthesis2,3,4,5,6,7 and exhibit susceptibility to ‘anti-PG’ antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We used a novel approach to metabolically label chlamydial PG using d-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis were labelled with these probes throughout their biphasic developmental life cycle, and the results of differential probe incorporation experiments conducted in the presence of ampicillin are consistent with the presence of chlamydial PG-modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence so far that chlamydial species possess functional PG.

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe’s developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host. Pathogenic Chlamydia do not assemble their peptidoglycan (PG) cell wall in a classical, mesh-like sacculus, but instead apparently confine it to the mid-cell in the actively dividing, non-infectious form. We characterize the assembly and aging of this PG-ring and link its synthesis to MreB, an actin-like protein associated with lateral cell wall synthesis in bacteria. As PG is recognized by the host innate immune system, we hypothesize that the limited amount of PG synthesized by Chlamydia is an adaptation to the microbe’s intracellular lifestyle.

Histone modifications are critical in regulating gene expression, cell cycle, cell proliferation, and development. Relatively few studies have investigated whether Helicobacter pylori, the major cause of human gastric diseases, affects histone modification. We therefore investigated the effects of H. pylori infection on histone modifications in a global and promoter-specific manner in gastric epithelial cells. Infection of gastric epithelial cells by wild-type H. pylori induced time- and dose-dependent dephosphorylation of histone H3 at serine 10 (H3 Ser10) and decreased acetylation of H3 lysine 23, but had no effects on seven other specific modifications. Different cag pathogenicity island (PAI)-containing-clinical isolates showed similar abilities to induce H3 Ser10 dephosphorylation. Mutation of cagA, vacA, nonphosphorylateable CagA mutant cagA_EPISA, or disruption of the flagella showed no effects, while deletion of the entire cagPAI restored the H3 Ser10 phosphorylation to control levels. Analysis of 27 cagPAI mutants indicated that the genes that caused H3 Ser10 dephosphorylation were similar to those that were previously found to induce interleukin-8, irrespective of CagA translocation. This effect was independent of ERK or p38 pathways and type I interferon signaling. Additionally, c-Jun and hsp70 gene expression was associated with this histone modification. These results demonstrate that H. pylori alters histone modification and host response via a cagA-, vacA-independent, but cagPAI-dependent mechanisms, which contribute to its persistent infection and pathogenesis.

Helicobacter pylori is a chronic colonizer of the gastric epithelium and plays a major role in the development of gastritis, peptic ulcer disease, and gastric cancer. In its coevolution with humans, the streamlining of the H. pylori genome has resulted in a significant reduction in metabolic pathways, one being purine nucleotide biosynthesis. Bioinformatic analysis has revealed that H. pylori lacks the enzymatic machinery for de novo production of IMP, the first purine nucleotide formed during GTP and ATP biosynthesis. This suggests that H. pylori must rely heavily on salvage of purines from the environment. In this study, we deleted several genes putatively involved in purine salvage and processing. The growth and survival of these mutants were analyzed in both nutrient-rich and minimal media, and the results confirmed the presence of a robust purine salvage pathway in H. pylori . Of the two phosphoribosyltransferase genes found in the H. pylori genome, only gpt appears to be essential, and an Δ apt mutant strain was still capable of growth on adenine, suggesting that adenine processing via Apt is not essential. Deletion of the putative nucleoside phosphorylase gene deoD resulted in an inability of H. pylori to grow on purine nucleosides or the purine base adenine. Our results suggest a purine requirement for growth of H. pylori in standard media, indicating that H. pylori possesses the ability to utilize purines and nucleosides from the environment in the absence of a de novo purine nucleotide biosynthesis pathway.

Inosine 5-monophosphate dehydrogenase (IMPDH) catalyzes the first committed step of guanosine 5-monophosphate (GMP) biosynthesis, and thus regulates the guanine nucleotide pool, which in turn governs proliferation. Human IMPDHs are validated targets for immunosuppressive, antiviral and anticancer drugs, but as yet microbial IMPDHs have not been exploited in antimicrobial chemotherapy. Selective inhibitors of IMPDH from Cryptosporidium parvum have recently been discovered that display anti-parasitic activity in cell culture models of infection. X-ray crystal structure and mutagenesis experiments identified the structural features that determine inhibitor susceptibility. These features are found in IMPDHs from a wide variety of pathogenic bacteria, including select agents and multiply drug resistant strains. A second generation inhibitor displays antibacterial activity against Helicobacter pylori, demonstrating the antibiotic potential of IMPDH inhibitors.

We describe the construction of mini-Tn 7 -based broad-host-range vectors encoding lux genes as bioluminescent reporters. These constructs can be mobilized into the desired host(s) by conjugation for chromosomal mini-Tn 7-lux integration and are useful for localization of bacteria during infections or for characterizing regulation of promoters of interest in Gram-negative bacteria.