lkyl Hydroperoxide Reductase Is Required for Helicobacter cinaedi Intestinal Colonization and Survival under Oxidative Stress in BALB / c and BALB / c Interleukin

19 Helicobacter cinaedi, a common human intestinal bacterium, has been implicated 20 in various enteric and systemic diseases in normal and immunocompromised patients. 21 Protection against oxidative stress is a crucial component of bacteria-host interactions. 22 Alkyl hydroperoxide reductase C (AhpC) is an enzyme responsible for detoxification of 23 peroxides and is important in protection from peroxide-induced stress. H. cinaedi 24 possesses a single ahpC, which was investigated with respect to its role in bacterial 25 survival during oxidative stress. The H. cinaedi ahpC mutant had diminished resistance 26 to organic hydroperoxide toxicity, but increased hydrogen peroxide resistance 27 compared with the wild type (WT) strain. The mutant also exhibited an oxygen-sensitive 28 phenotype and was more susceptible to killing by macrophages than the WT strain. In 29 vivo experiments in BALB/c and BALB/c IL10-/mice revealed that the cecal colonizing 30 ability of the ahpC mutant was significantly reduced. The mutant also had diminished 31 ability to induce bacterial-specific immune responses in vivo, as shown by 32 immunoglobulin (IgG2a and IgG1) serum levels. Collectively, these data suggest that 33 H. cinaedi ahpC not only contributes to protecting the organism against oxidative stress 34 but also alters its pathogenic properties in vivo. 35 Introduction 36 Historically, Helicobacter cinaedi was classified under the genus Campylobacter; 37 however, it was subsequently classified as a Helicobacter sp. based on DNA-DNA 38 hybridization, 16S rRNA analysis, and biochemical properties (36). H. cinaedi has been 39 reported by Vandamme P. et al. to form a 16S rRNA taxonomic cluster with H. canis, H. 40 bilis, and Flexispira rappini, separate from the H. pylori cluster (42). H. cinaedi is now 41 recognized as an enterohepatic helicobacter colonizing the lower gastrointestinal tract 42 of numerous mammals, including dogs, cats, hamsters, and monkeys (12). Although the 43 epidemiology and pathogenesis of H. cinaedi infections are not fully elucidated, it was 44 first isolated from rectal swabs obtained from homosexual men (40). It is also 45 implicated as a cause of gastroenteritis, particularly in immunocompromised individuals, 46 such as HIV-infected or cancer patients, and recently was isolated from a healthy 47 heterosexual male with cellulitis (16). Unlike some other Helicobacter spp. and 48 Campylobacter spp. -related organisms, which colonize the intestinal tract (36), H. 49 cinaedi has been cultured from the blood of patients with sepsis (16, 20, 23) and can 50 cause cellulitis, bacteremia, and gastroenteritis with a high potential for recurrence (38). 51 In general, innate immunity is programmed to respond immediately when a host 52 is challenged by an infectious pathogen, whereas adaptive immunity, mounted in 53 response to infection, requires time to react and generate a microbe-specific response. 54 One of the primary defense mechanisms of the innate response is macrophage killing, 55 in which activated macrophages produce various reactive oxygen species (ROS), 56 including organic hydroperoxides. These compounds cause damage to DNA, RNA, 57 protein, and lipids of invading microorganisms. In response, bacterial pathogens have 58 developed both non-enzymatic and enzymatic mechanisms to protect themselves from 59 damage and facilitate successful resistance to macrophage killing. An important 60 example of this microbial defense mechanism is the enzyme alkyl hydroperoxide 61 reductase C (AhpC), which catalyzes the hydrolysis of toxic compounds such as organic 62 hydroperoxide to the corresponding alcohol and water. AhpC is classified as a member 63 of the peroxiredoxin (Prx) family because it contains the CXXC motif, a common feature 64 of Prx-type peroxidases (9). Its peroxidatic cysteine reacts with peroxides to yield the 65 corresponding alcohol and cysteine sulfenic acid (Cys-SOH), which is then reduced by 66 the free thiol of the cysteine residue to form a disulfide bond to complete the catalytic 67 cycle. Reflecting its importance in protecting organisms against oxidative stress, ahpC 68 has been identified in a wide variety of eubacteria and archaea. We therefore 69 hypothesized that it contributes to the survival of H. cinaedi during infection, and not 70 only plays an important role in colonization, but also in potential virulence. In vitro and in 71 vivo studies were performed to assess the oxidative stress response of WT H. cinaedi 72 and isogenic mutants lacking ahpC. 73 74 Material and Methods 75 Bacterial strains and growth conditions. H. cinaedi (CCUG18818) and E. coli DH5a 76 were used for genetic manipulations. H. cinaedi was grown on Tryptic Soy Agar (TSA) 77 supplemented with 10% sheep’s blood) or brucella broth (BB) supplemented with 10% 78 fetal calf serum; 25 μg/ml of chloramphenicol was added as appropriate. Plates were 79 grown microaerobically at 37C in an incubator with 10% CO2, 10% H2, and 80% N2 for 80 3 to 5 days. E. coli was grown in Luria-Bertani (LB) media supplemented with 100 81 μg/ml of ampicillin, or 30 μg/ml carbenicillin and incubated aerobically at 37C (13). 82 Construction of H. cinaedi ahpC mutant strain by insertional mutagenesis. Briefly, 83 the ahpC gene was PCR amplified from H. cinaedi chromosomal DNA using primers 84 encompassing a SmaI restriction site in the middle of the gene. The products were 85 ligated into pGemTeasy Vector (Promega Madison, WI) and transformed into E. coli 86 DH5a, generating the plasmid pGemTeasy-ahpC. It was digested by SmaI and ligated 87 to a chloramphenicol cassette that was cut by HincII from pUC20CAT. The 88 pGemTeasy-ahpC::CAT was transformed into the H. cinaedi parental strain by 89 electroporation facilitating a double crossover event at the flanking regions, resulting in 90 inactivation of ahpC gene. The chloramphenicol resistant clones were selected, and the 91 presence of the ahpC mutation was verified by PCR and sequencing. Mutants were 92 confirmed by Southern blot analysis, as follows. Genomic DNA was digested by HindIII, 93 separated on 1% agarose gel, transferred to a membrane, and hybridized with probes 94 (amplified by using NC5; 5’ ATATGTTAGTTACAAAACTTGC 3’ and NC8; 5’ 95 ATTAAAGCTTAATGGAATTTTCT 3’). HindIII digestion of H. cinaedi genomic DNA 96 produces a 1210-bp positive hybridization band, whereas the integration of pUC20CAT 97 (2.0 kb) into the gene results in one large band: 2010-bp. As shuttle vectors are not 98 available for H. cinaedi, an independent ahpC mutant was constructed and its 99 phenotype tested to confirm that the altered phenotype arose from ahpC inactivation, 100 not from random mutations. 101