Sustained Helicobacter pylori infection accelerates gastric dysplasia in a mouse model.

More than 80% of gastric cancer is attributable to stomach infection with Helicobacter pylori (Hp). Gastric preneoplastic progression involves sequential tissue changes, including loss of parietal cells, metaplasia and dysplasia. In transgenic mice, active KRAS expression recapitulates these tissue changes in the absence of Hp infection. This model provides an experimental system to investigate additional roles of Hp in preneoplastic progression, beyond its known role in initiating inflammation. Tissue histology, gene expression, the immune cell repertoire, and metaplasia and dysplasia marker expression were assessed in KRAS+ mice +/-Hp infection. Hp+/KRAS+ mice had severe T-cell infiltration and altered macrophage polarization; a different trajectory of metaplasia; more dysplastic glands; and greater proliferation of metaplastic and dysplastic glands. Eradication of Hp with antibiotics, even after onset of metaplasia, prevented or reversed these tissue phenotypes. These results suggest that gastric preneoplastic progression differs between Hp+ and Hp- cases, and that sustained Hp infection can promote the later stages of gastric preneoplastic progression.

A Genome-Wide Helicobacter pylori Morphology Screen Uncovers a Membrane-Spanning Helical Cell Shape Complex.

Evident in its name, the gastric pathogen Helicobacter pylori has a helical cell morphology which facilitates efficient colonization of the human stomach. An improved light-focusing strategy allowed us to robustly distinguish even subtle perturbations of H. pylori cell morphology by deviations in light-scattering properties measured by flow cytometry. Profiling of an arrayed genome-wide deletion library identified 28 genes that influence different aspects of cell shape, including properties of the helix, cell length or width, cell filament formation, cell shape heterogeneity, and cell branching. Included in this mutant collection were two that failed to form any helical cells, a soluble lytic transglycosylase and a previously uncharacterized putative multipass inner membrane protein HPG27_0728, renamed Csd7. A combination of cell fractionation, mutational, and immunoprecipitation experiments show that Csd7 and Csd2 collaborate to stabilize the Csd1 peptidoglycan (PG) endopeptidase. Thus, both csd2 and csd7 mutants show the same enhancement of PG tetra-pentapeptide cross-linking as csd1 mutants. Csd7 also links Csd1 with the bactofilin CcmA via protein-protein interactions. Although Csd1 is stable in ccmA mutants, these mutants show altered PG tetra-pentapeptide cross-linking, suggesting that Csd7 may directly or indirectly activate as well as stabilize Csd1. These data begin to illuminate a highly orchestrated program to regulate PG modifications that promote helical shape, which includes nine nonessential nonredundant genes required for helical shape and 26 additional genes that further modify H. pylori's cell morphology.IMPORTANCE The stomach ulcer and cancer-causing pathogen Helicobacter pylori has a helical cell shape which facilitates stomach infection. Using light scattering to measure perturbations of cell morphology, we identified 28 genes that influence different aspects of cell shape. A mutant in a previously uncharacterized protein renamed Csd7 failed to form any helical cells. Biochemical analyses showed that Csd7 collaborates with other proteins to stabilize the cell wall-degrading enzyme Csd1. Csd7 also links Csd1 with a putative filament-forming protein via protein-protein interactions. These data suggest that helical cell shape arises from a highly orchestrated program to regulate cell wall modifications. Targeting of this helical cell shape-promoting program could offer new ways to block infectivity of this important human pathogen.

Nonhelical Helicobacter pylori Mutants Show Altered Gland Colonization and Elicit Less Gastric Pathology than Helical Bacteria during Chronic Infection.

Half of all humans harbor Helicobacter pylori in their stomachs. Helical cell shape is thought to facilitate H. pylori's ability to bore into the protective mucus layer in a corkscrew-like motion, thereby enhancing colonization of the stomach. H. pylori cell shape mutants show impaired colonization of the mouse stomach, highlighting the importance of cell shape in infection. To gain a deeper understanding of how helical cell morphology promotes host colonization by H. pylori, we used three-dimensional confocal microscopy to visualize the clinical isolate PMSS1 and an isogenic straight-rod mutant (Δcsd6) within thick longitudinal mouse stomach sections. We also performed volumetric image analysis to quantify the number of bacteria residing within corpus and antral glands in addition to measuring total CFU. We found that straight rods show attenuation during acute colonization of the stomach (1 day or 1 week postinfection) as measured by total CFU. Our quantitative imaging revealed that wild-type bacteria extensively colonized antral glands at 1 week postinfection, while csd6 mutants showed variable colonization of the antrum at this time point. During chronic infection (1 or 3 months postinfection), total CFU were highly variable but similar for wild-type and straight rods. Both wild-type and straight rods persisted and expanded in corpus glands during chronic infection. However, the straight rods showed reduced inflammation and disease progression. Thus, helical cell shape contributes to tissue interactions that promote inflammation during chronic infection, in addition to facilitating niche acquisition during acute infection.

Increased H. pylori stool shedding and EPIYA-D cagA alleles are associated with gastric cancer in an East Asian hospital.

BACKGROUND: Helicobacter pylori infection increases risk for gastric cancer. Geographic variation in gastric cancer risk has been attributed to variation in carriage and type of the H. pylori oncogene cagA. Colonization density may also influence disease and cagA has been associated with higher shedding in stool. However, the relationship between H. pylori load in the stool and in the stomach is not clear. METHODS: To investigate possible differences in H. pylori load in the stomach and shedding in stool, H. pylori load and cagA genotype were assessed using droplet digital PCR assays on gastric mucosa and stool samples from 49 urea breath test-positive individuals, including 25 gastric cancer and 24 non-cancer subjects at Henan Cancer Hospital, Henan, China. RESULTS: Quantitation of H. pylori DNA indicated similar gastric loads among cancer and non-cancer cases, but the gastric cancer group had a median H. pylori load in the stool that was six times higher than that of the non-cancer subjects. While the cagA gene was uniformly present among study subjects, only 70% had the East Asian cagA allele, which was significantly associated with gastric cancer (Fisher's Exact Test, p = 0.03). CONCLUSION: H. pylori persists in a subset of gastric cancer cases and thus may contribute to cancer progression. In this East Asian population with a high prevalence of the cagA gene, the East Asian allele could still provide a marker for gastric cancer risk. IMPACT: This study contributes to our understanding of H. pylori dynamics in the context of pathological changes.

High prevalence of Helicobacter pylori clarithromycin resistance mutations among Seattle patients measured by droplet digital PCR.

BACKGROUND: Treatment of Helicobacter pylori infection is often empiric; however, current guidelines for management of Helicobacter pylori infection advise against the use of standard triple therapy (clarithromycin, amoxicillin, and proton-pump inhibitor) when clarithromycin resistance exceeds 20%. We developed and tested a new culture-free assay to detect clarithromycin resistance-conferring mutations to determine the prevalence of H. pylori clarithromycin resistance in patients from the United States Pacific Northwest. MATERIALS AND METHODS: Droplet digital PCR (ddPCR) was used to detect the H. pylori 23S rRNA gene, and resistance-conferring mutations, in archived, formalin-fixed, paraffin-embedded (FFPE) gastric tissue and to retrospectively determine the prevalence of clarithromycin-resistant H. pylori among 110 patients at an academic medical center in the Northwest United States between 2012 and 2014. RESULTS: Of 102 patients with the H. pylori 23S rRNA gene detected by the ddPCR assay, 45 (44%) had clarithromycin resistance mutations. Thirty-three of the 45 patients with clarithromycin resistance mutations had a mix of wild-type and resistance alleles. Prevalence of clarithromycin resistance mutations differed among racial groups and was highest among Asians, with mutations detected in 14 (67%) of the 21 patient samples. CONCLUSIONS: The prevalence of clarithromycin resistance detected in this region exceeds 20%, indicating that standard triple therapy should not be the first-line antibiotic treatment for H. pylori infection. Culture-free assays for detecting clarithromycin resistance mutations can be performed on archived tissue samples and will aid in informing tailored treatment for effective H. pylori eradication.

Identification of long-lived proteins retained in cells undergoing repeated asymmetric divisions.

Long-lived proteins have been implicated in age-associated decline in metazoa, but they have only been identified in extracellular matrices or postmitotic cells. However, the aging process also occurs in dividing cells undergoing repeated asymmetric divisions. It was not clear whether long-lived proteins exist in asymmetrically dividing cells or whether they are involved in aging. Here we identify long-lived proteins in dividing cells during aging using the budding yeast, Saccharomyces cerevisiae. Yeast mother cells undergo a limited number of asymmetric divisions that define replicative lifespan. We used stable-isotope pulse-chase and total proteome mass-spectrometry to identify proteins that were both long-lived and retained in aging mother cells after ∼ 18 cells divisions. We identified ∼ 135 proteins that we designate as long-lived asymmetrically retained proteins (LARPS). Surprisingly, the majority of LARPs appeared to be stable fragments of their original full-length protein. However, 15% of LARPs were full-length proteins and we confirmed several candidates to be long-lived and retained in mother cells by time-lapse microscopy. Some LARPs localized to the plasma membrane and remained robustly in the mother cell upon cell division. Other full-length LARPs were assembled into large cytoplasmic structures that had a strong bias to remain in mother cells. We identified age-associated changes to LARPs that include an increase in their levels during aging because of their continued synthesis, which is not balanced by turnover. Additionally, several LARPs were posttranslationally modified during aging. We suggest that LARPs contribute to age-associated phenotypes and likely exist in other organisms.

Replicative age induces mitotic recombination in the ribosomal RNA gene cluster of Saccharomyces cerevisiae.

Somatic mutations contribute to the development of age-associated disease. In earlier work, we found that, at high frequency, aging Saccharomyces cerevisiae diploid cells produce daughters without mitochondrial DNA, leading to loss of respiration competence and increased loss of heterozygosity (LOH) in the nuclear genome. Here we used the recently developed Mother Enrichment Program to ask whether aging cells that maintain the ability to produce respiration-competent daughters also experience increased genomic instability. We discovered that this population exhibits a distinct genomic instability phenotype that primarily affects the repeated ribosomal RNA gene array (rDNA array). As diploid cells passed their median replicative life span, recombination rates between rDNA arrays on homologous chromosomes progressively increased, resulting in mutational events that generated LOH at >300 contiguous open reading frames on the right arm of chromosome XII. We show that, while these recombination events were dependent on the replication fork block protein Fob1, the aging process that underlies this phenotype is Fob1-independent. Furthermore, we provide evidence that this aging process is not driven by mechanisms that modulate rDNA recombination in young cells, including loss of cohesion within the rDNA array or loss of Sir2 function. Instead, we suggest that the age-associated increase in rDNA recombination is a response to increasing DNA replication stress generated in aging cells.

Tick-borne transmission of two genetically distinct Anaplasma marginale strains following superinfection of the mammalian reservoir host.

Strain superinfection affects the dynamics of epidemiological spread of pathogens through a host population. Superinfection has recently been shown to occur for two genetically distinct strains of the tick-borne pathogen Anaplasma marginale that encode distinctly different surface protein variants. Superinfected animals could serve as a reservoir for onward transmission of both strains if the tick vector is capable of acquiring and transmitting both strains. Whether competition among strains during development within the tick vector, which requires sequential invasion and replication events, limits colonization and subsequent transmission to a single strain is unknown. We tested this possibility by acquisition feeding Dermacentor andersoni ticks on a reservoir host superinfected with the genetically distinct St. Maries and EMPhi strains. Although the St. Maries strain consistently maintained higher bacteremia levels in the mammalian host and the EMPhi strain had an early advantage in colonization of the tick salivary glands, individual ticks were coinfected, and there was successful transmission of both strains. These results indicate that a genetically distinct A. marginale strain capable of superinfecting the mammalian host can subsequently be cotransmitted and become established within the host population despite the presence of an existing established strain.

Selection for simple major surface protein 2 variants during Anaplasma marginale transmission to immunologically naïve animals.

Anaplasma marginale, a rickettsial pathogen, evades clearance in the animal host by antigenic variation. Under immune selection, A. marginale expresses complex major surface protein 2 mosaics, derived from multiple donor sequences. However, these mosaics have a selective advantage only in the presence of adaptive immunity and are rapidly replaced by simple variants following transmission.