Colonization of the human stomach by Helicobacter pylori is an important risk factor for development of gastric cancer. The H. pylori cag pathogenicity island (cag PAI) encodes components of a type IV secretion system (T4SS) that translocates the bacterial oncoprotein CagA into gastric epithelial cells, and CagL is a specialized component of the cag T4SS that binds the host receptor α5β1 integrin. Here, we utilized a mass spectrometry-based approach to reveal co-purification of CagL, CagI (another integrin-binding protein), and CagH (a protein with weak sequence similarity to CagL). These three proteins are encoded by contiguous genes in the cag PAI, and are detectable on the bacterial surface. All three proteins are required for CagA translocation into host cells and H. pylori-induced IL-8 secretion by gastric epithelial cells; however, these proteins are not homologous to components of T4SSs in other bacterial species. Scanning electron microscopy analysis reveals that these proteins are involved in the formation of pili at the interface between H. pylori and gastric epithelial cells. ΔcagI and ΔcagL mutant strains fail to form pili, whereas a ΔcagH mutant strain exhibits a hyperpiliated phenotype and produces pili that are elongated and thickened compared to those of the wild-type strain. This suggests that pilus dimensions are regulated by CagH. A conserved C-terminal hexapeptide motif is present in CagH, CagI, and CagL. Deletion of these motifs results in abrogation of CagA translocation and IL-8 induction, and the C-terminal motifs of CagI and CagL are required for formation of pili. In summary, these results indicate that CagH, CagI, and CagL are components of a T4SS subassembly involved in pilus biogenesis, and highlight the important role played by unique constituents of the H. pylori cag T4SS. Helicobacter pylori persistently colonizes the stomach in approximately half of the human population. People who are infected with H. pylori strains harboring the cag pathogenicity island (PAI) have an increased risk of developing gastric cancer. The cag PAI encodes a type IV secretion system (T4SS) that is utilized by the bacteria to inject the bacterial oncoprotein CagA into gastric epithelial cells. Related T4SSs found in several other bacteria have been studied in detail, but thus far there has been very little study of the H. pylori cag T4SS. Here, we utilized a mass spectrometry-based approach to reveal co-purification of three constituents of the H. pylori T4SS (CagH, CagI, and CagL) that lack homology to components of T4SSs in other bacterial species. These proteins are essential for CagA translocation into host cells, and scanning electron microscope studies reveal that the proteins are involved in the formation of pili at the bacterial-host cell interface. A conserved C-terminal motif present in CagH, CagI, and CagL is essential for functionality of the T4SS. This study highlights the important role played by unique constituents of the H. pylori cag T4SS, and illustrates the marked variation that exists among bacterial T4SSs.

Oxidative stress (OS) is a condition that occurs as a result of an imbalance between the production of reactive oxygen species (ROS) and the body’s ability to detoxify and neutralize them. It can play a role in a variety of reproductive system conditions, including polycystic ovary syndrome (PCOS), endometriosis, preeclampsia, and infertility. In this review, we briefly discuss the links between oxidative stress and PCOS. Mitochondrial mutations may lead to impaired oxidative phosphorylation (OXPHOS), decreased adenosine triphosphate (ATP) production, and an increased production of ROS. These functional consequences may contribute to the metabolic and hormonal dysregulation observed in PCOS. Studies have shown that OS negatively affects ovarian follicles and disrupts normal follicular development and maturation. Excessive ROS may damage oocytes and granulosa cells within the follicles, impairing their quality and compromising fertility. Impaired OXPHOS and mitochondrial dysfunction may contribute to insulin resistance (IR) by disrupting insulin signaling pathways and impairing glucose metabolism. Due to dysfunctional OXPHOS, reduced ATP production, may hinder insulin-stimulated glucose uptake, leading to IR. Hyperandrogenism promotes inflammation and IR, both of which can increase the production of ROS and lead to OS. A detrimental feedback loop ensues as IR escalates, causing elevated insulin levels that exacerbate OS. Exploring the relations between OS and PCOS is crucial to fully understand the role of OS in the pathophysiology of PCOS and to develop effective treatment strategies to improve the quality of life of women affected by this condition. The role of antioxidants as potential therapies is also discussed.

Helicobacter pylori causes numerous alterations in gastric epithelial cells through processes that are dependent on activity of the cag type IV secretion system (T4SS). Filamentous structures termed “pili” have been visualized at the interface between H. pylori and gastric epithelial cells, and previous studies suggested that pilus formation is dependent on the presence of the cag pathogenicity island (PAI). Thus far, there has been relatively little effort to identify specific genes that are required for pilus formation, and the role of pili in T4SS function is unclear. In this study, we selected 7 genes in the cag PAI that are known to be required for T4SS function and investigated whether these genes were required for pilus formation. cagT , cagX , cagV , cagM , and cag3 mutants were defective in both T4SS function and pilus formation; complemented mutants regained T4SS function and the capacity for pilus formation. cagY and cagC mutants were defective in T4SS function but retained the capacity for pilus formation. These results define a set of cag PAI genes that are required for both pilus biogenesis and T4SS function and reveal that these processes can be uncoupled in specific mutant strains.

The BabA adhesin of Helicobacter pylori is an outer membrane protein that binds to the fucosylated Lewis b histo-blood group antigen on the surface of gastric epithelial cells. We screened a phage-displayed ScFv (single-chain fragment variable) recombinant antibody library for antibodies reactive with a recombinant BabA fragment and identified two such antibodies. Each antibody recognized an ∼75-kDa protein present in wild-type H. pylori strain J99 but absent from an isogenic babA mutant strain. An immunoreactive BabA protein was detected by at least one of the antibodies in 18 (46%) of 39 different wild-type H. pylori strains and was detected more commonly in cagA-positive strains than in cagA-negative strains. Numerous amino acid polymorphisms were detected among BabA proteins expressed by different strains, with the greatest diversity occurring in the middle region of the proteins. Among the 18 strains that expressed a detectable BabA protein, there was considerable variation in the level of binding to Lewis b in vitro. Heterogeneity among H. pylori strains in expression of the BabA protein may be a factor that contributes to differing clinical outcomes among H. pylori-infected humans.

Earlier studies from our laboratories characterized the mutation profile of the optically active (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-BPDE—the ultimate carcinogenic metabolite of benzo[a]pyrene] in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene of Chinese hamster V-79 cells. In the present study, we evaluated the mutation profile of (−)-7S,8R-dihydroxy-9R, 10S-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(−)-BPDE—a weakly carcinogenic or inactive enantiomer] and compared its mutation profile with that of (+)-BPDE. In both diol epoxide enantiomers, the benzylic 7-hydroxy group and epoxide oxygen are trans. The mutation frequency for V-79 cells treated with DMSO vehicle or with a low, non-cytotoxic dose (0.5 μM) or a high cytotoxic dose (2.0 μM) of (−)-BPDE was 1, 25 or 185 8-azaguanine-resistant colonies/10⁵ survivors, respectively. Independent 8-azaguanine-resistant clones were isolated, and complementary DNAs were prepared by reverse transcription. The coding region of the HPRT gene was amplified by the polymerase chain reaction and sequenced. Altogether, 92 (−)-BPDE-induced mutant clones were examined. At both doses, base substitutions were the most prevalent mutations observed (present in −;70% of the mutant clones), followed by exon deletions (present in −22% of the mutant clones) and frame shift mutations (present in −6% of the mutant clones) in the cDNAs analyzed. At the high cytotoxic dose, 5 out of 36 base substitutions occurred at AT base pairs (14%) and 31 at GC base pairs (86%). At the low, non-cytotoxic dose, 7 out of 34 base substitutions were at AT base pairs (21%) and 27 were at GC base pairs (79%). Although there was a trend towards an increase in the proportion of mutations at AT base pairs when the dose of (−)-BPDE was decreased, this trend was not statistically significant. The data also indicated no dose-dependent differences in the kinds of base substitutions or exon deletions in cDNAs induced by (−)-BPDE. Ninety-one per cent of the (−)-BPDE-induced mutations that occurred at guanine were on the non-transcribed strand of DNA and 9% were on the transcribed strand. In contrast to these results, 50% of the (−)-BPDE-induced mutations that occurred at adenine were on the transcribed strand and 50− on the non-transcribed strand. A comparison of the mutation profiles of (−)- and (+)-BPDE revealed that (i) (+)-BPDE is more mutagenic than (−)-BPDE, (ii) (+)-BPDE caused a significantly higher proportion of mutations at AT base pairs as the dose decreased, but this was not observed for (−)-BPDE, (iii) (+)-BPDE showed high selectivity for GC → TA transversions, but considerably less selectivity was observed for (−)-BPDE, and (iv) (−)-BPDE had different hot spots for base substitutions than did (+)-BPDE.

Mutations in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (HPRT) gene of Chinese hamster V-79 cells were examined after exposure of the cells to a high cytotoxic dose (0.48 microM; 35% survival) and a low noncytotoxic dose (0.04 microM; 100% survival) of the ultimate carcinogen (+)-7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-BPDE]. Independent 8-azaguanine-resistant colonies were isolated and cDNAs were prepared by reverse transcription. The coding region of the cDNA of the HPRT gene was amplified by the polymerase chain reaction and sequenced. An examination of the DNA base sequence changes induced by different doses of (+)-BPDE demonstrated that the high dose of (+)-BPDE caused base substitution mutations almost exclusively at G.C base pairs whereas the low dose of (+)-BPDE caused mutations at both G.C and A.T base pairs. Thus, use of a low dose of (+)-BPDE allowed the detection of mutations (at A.T base pairs) that were not readily observed with a high dose of (+)-BPDE. The data also suggest that the low dose of (+)-BPDE may have caused a different profile of base substitutions at G.C base pairs and exon deletions than the high dose. The results indicate dose-dependent differences in the profile of mutations for an ultimate carcinogen.