HPAA

CAS No.: 23783-26-8
EC No.: 405-710-8

Synonyms:
HPAA; hpaa; Acetic acid, 2-hydroxy-2-phosphono- [ACD/Index Name]; Acetic acid, hydroxyphosphono-; Acide hydroxy(phosphono)acétique [French] [ACD/IUPAC Name]; Hydroxy(phosphono)acetic acid [ACD/IUPAC Name]; Hydroxy(phosphono)essigsäure [German] [ACD/IUPAC Name]; HPAA; hpaa; HPAA; hpaa; 115469-15-3 [RN]; 153733-51-8 [RN]; 23783-26-8 [RN]; 2-Hydroxy phosphono acetic acid; 2-Hydroxy-2-phosphonoacetic acid; 2-hydroxyphosphonoacetic acid; 2-Hydroxyphosphonocarboxylic Acid; HPAA; hpaa; HPAA; hpaa; 2-phosphoglycolic acid; ACETIC ACID 2-HYDROXY-2-PHOSPHONO-; HPAA; Hydroxyphosphono-acetic acid; Hydroxyphosphono-acetic acid|2-Hydroxy phosphono acetic acid|HPAA; MFCD01940970; MFCD20638215; Phosphoglycolic acid; PHOSPHONOGLYCOLIC ACID; α-Hydroxyl phosphonoacetic acid; HPAA; hpaa; HPAA; hpaa; α-Hydroxyphosphonoacetic acid; 2-Hydroxy-2-phosphono-acetic acid; 2-hydroxy-2-phosphonoacetic acid; 2-hydroxy-2-phosphono-ethanoic acid; Acetic acid, hydroxyphosphono-; alpha.-Hydroxyl phosphonoacetic acid; alpha.-Hydroxyphosphonoacetic acid; HPA; HPAA; HPSE; HPSE1; Belcor 575; Heparanase-1; HPAA; hpaa; HPAA; hpaa; Snailagglutinin; Endo-glucoronidase; Phosphonoglycolic acid; Heparanase [Precursor]; Hydroxyphosphonoacetic acid; Acetic acid, hydroxyphosphono-; 2-[hydroperoxy(hydroxy)phosphoryl]acetic acid; 2-(Hydroperoxy(hydroxy)phosphoryl)acetic acid; hydroxyphosphono acetic acid; HPAA; hpaa; HPAA; hpaa; SCHEMBL560738; ZINC100443591; DB-029902; Hydroxyphosphono-acetic acid; 2-Hydroxy phosphono acetic acid; HPA; HPAA; HPAA; hpaa; HPAA; hpaa; Hydroxyphosphono-acetic acid; N-HEXYLPHOSPHONIC ACID; LABOTEST-BB LT00408920; hpaa; n-Hexylphosphonicacid,min.97%; 2-Hydroxyphosphonocarboxylic Acid; 2-Hydroxy Phosphonoacetic Acid; 2-Hydroxy Phosphonic Acetic AcidStructure; hexylphosphonic acid; hexylphosphonate; Acetic acid, 2-hydroxy-2-phosphono-; 2-hydroxy-2-phosphonoacetic acid; HPAA; hpaa; HPAA; hpaa; 2-Hydroxyphosphonoacetic Acid; 2-hydroxy-2-phosphono-acetic acid; Phosphono-hydroxy-acetic acid; SCHEMBL254667; 2-Hydroxyphosphonocarboxylic Acid; CTK1A2951; DTXSID40865115; .alpha.-Hydroxyphosphonoacetic acid; HPAA; hpaa; HPAA; hpaa; .alpha.-Hydroxyl phosphonoacetic acid; AKOS015892863; VZ34695; FT-0694552; EC 405-710-8; 2-Hydroxy-2-phosphonoacetic acid 23783-26-8; HPAA; hpaa; HPAA; hpaa; Acetic acid, 2-hydroxy-2-phosphono- [ACD/Index Name]; Acetic acid, hydroxyphosphono-; Acide hydroxy(phosphono)acétique [French] [ACD/IUPAC Name]; Hydroxy(phosphono)acetic acid [ACD/IUPAC Name]; Hydroxy(phosphono)essigsäure [German] [ACD/IUPAC Name]; HPAA; hpaa; HPAA; hpaa; 115469-15-3 [RN]; 153733-51-8 [RN]; 23783-26-8 [RN]; 2-Hydroxy phosphono acetic acid; 2-Hydroxy-2-phosphonoacetic acid; 2-hydroxyphosphonoacetic acid; 2-Hydroxyphosphonocarboxylic Acid; HPAA; hpaa; HPAA; hpaa; 2-phosphoglycolic acid; ACETIC ACID 2-HYDROXY-2-PHOSPHONO-; HPAA; Hydroxyphosphono-acetic acid; Hydroxyphosphono-acetic acid|2-Hydroxy phosphono acetic acid|HPAA; MFCD01940970; MFCD20638215; Phosphoglycolic acid; PHOSPHONOGLYCOLIC ACID; α-Hydroxyl phosphonoacetic acid; HPAA; hpaa; HPAA; hpaa; α-Hydroxyphosphonoacetic acid; 2-Hydroxy-2-phosphono-acetic acid; 2-hydroxy-2-phosphonoacetic acid; 2-hydroxy-2-phosphono-ethanoic acid; Acetic acid, hydroxyphosphono-; alpha.-Hydroxyl phosphonoacetic acid; alpha.-Hydroxyphosphonoacetic acid; HPA; HPAA; HPSE; HPSE1; Belcor 575; Heparanase-1; HPAA; hpaa; HPAA; hpaa; Snailagglutinin; Endo-glucoronidase; Phosphonoglycolic acid; Heparanase [Precursor]; Hydroxyphosphonoacetic acid; Acetic acid, hydroxyphosphono-; 2-[hydroperoxy(hydroxy)phosphoryl]acetic acid; 2-(Hydroperoxy(hydroxy)phosphoryl)acetic acid; hydroxyphosphono acetic acid; HPAA; hpaa; HPAA; hpaa; SCHEMBL560738; ZINC100443591; DB-029902; Hydroxyphosphono-acetic acid; 2-Hydroxy phosphono acetic acid; HPA; HPAA; HPAA; hpaa; HPAA; hpaa; Hydroxyphosphono-acetic acid; N-HEXYLPHOSPHONIC ACID; LABOTEST-BB LT00408920; hpaa; n-Hexylphosphonicacid,min.97%; 2-Hydroxyphosphonocarboxylic Acid; 2-Hydroxy Phosphonoacetic Acid; 2-Hydroxy Phosphonic Acetic AcidStructure; hexylphosphonic acid; hexylphosphonate; Acetic acid, 2-hydroxy-2-phosphono-; 2-hydroxy-2-phosphonoacetic acid; HPAA; hpaa; HPAA; hpaa; 2-Hydroxyphosphonoacetic Acid; 2-hydroxy-2-phosphono-acetic acid; Phosphono-hydroxy-acetic acid; SCHEMBL254667; 2-Hydroxyphosphonocarboxylic Acid; CTK1A2951; DTXSID40865115; .alpha.-Hydroxyphosphonoacetic acid; HPAA; hpaa; HPAA; hpaa; .alpha.-Hydroxyl phosphonoacetic acid; AKOS015892863; VZ34695; FT-0694552; EC 405-710-8; 2-Hydroxy-2-phosphonoacetic acid 23783-26-8; HPAA; hpaa

HPAA

ABSTRACT
Infection with the human gastric pathogen Helicobacter pylori can give rise to chronic gastritis, peptic ulcer, and gastric cancer. All H. pylori strains express the surface-localized protein HpaA, a promising candidate for a vaccine against H. pylori infection. To study the physiological importance of HpaA, a mutation of the hpaA gene was introduced into a mouse-adapted H. pylori strain. To justify that the interruption of the hpaA gene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavily H. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization of H. pylori in mice.

H. pylori adhesin A (HpaA) is a surface-located (7, 14, 20) lipoprotein (25) that was initially described as a sialic acid binding adhesin, but supportive evidence is still lacking. It is recognized by antibodies from H. pylori-infected individuals (23, 39), and the expression of the HpaA protein has previously been found to be highly conserved among H. pylori isolates (9, 39). Furthermore, genomic studies (2, 32) show no significant sequence homologies of HpaA with other known proteins. Taken together, this makes HpaA a putative candidate as a vaccine antigen against H. pylori infection.

In this study, we have constructed an HpaA mutant in the mouse-adapted H. pylori Sydney strain 1 (SS1) to examine the role of HpaA in colonization. Because of cotranscription, constructed gene mutations have the potential to cause polar effects, i.e., inhibiting expression of downstream genes in an operon. In addition, it has been shown that knocking out one gene can affect other genes in an unpredicted manner (30). Thus, when studying a mutant, proteomic analysis offers a convenient method to monitor changes in protein expression without prior knowledge of what those changes might be.

The first aim of this study was to examine the overall protein profile, including the protein expression of the genes located downstream of hpaA, of the mouse-adapted SS1 strain and its isogenic HpaA mutant. This was achieved by a proteomic approach where whole-cell extracts of the bacteria were compared by DIGE analysis. We also combined subcellular fractionation and one-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis with nano-LC Fourier transform (FT) ion cyclotron resonance (ICR) (FT-ICR) MS and tandem MS (MS/MS) analyses in order to compare the OMP profiles of the SS1 wild-type and mutant strains. To determine whether HpaA is essential for survival in the host, mice were infected with either H. pylori SS1 or the HpaA mutant strain, and the colonization levels

MATERIALS AND METHODS
Construction of SS1 hpaA-negative/deficient mutant SS1(ΔhpaA).
The hpaA mutant was originally constructed in H. pylori strain CCUG 17874 by a two-step amplification resulting in a 450-bp deletion of the hpaA gene (kindly provided by P. Doig et al., Astrazeneca Research Centre, Boston, MA) and insertion of a 1.4-kb kanamycin cassette (25). The mutation was transferred from H. pylori CCUG 17874 to the mouse-adapted SS1 strain by natural transformation. Five kanamycin-resistant transformants were analyzed by PCR with two HpaA-specific primers (forward primer, 5′-GGCGTAGAAATGGAAGCG-3′; reverse primer, 5′-CCCAAGCTTCATCAGCCCTTAAATACACG-3′) (21) to confirm that the kanamycin cassette was inserted in the hpaA gene, resulting in a larger PCR product than of that of the wild-type SS1 strain. One of the transformants with the correct insertion was further characterized by SDS-PAGE and immunoblotting with the monoclonal antibody HP30-1:1:6, specific for HpaA (9). This strain, SS1(ΔhpaA), was negative in the immunoblot.

Strains and culture conditions.
The mouse-adapted H. pylori strains SS1 (CagA+ VacA+ Ley) (19) and SS1(ΔhpaA) were used in all experiments and stored at −70°C as stock cultures. For preparation of antigens from SS1 and SS1(ΔhpaA), the bacteria were grown on Colombia-Iso agar plates to confluence for 3 days under microaerophilic conditions (10% CO2, 6% O2, and 84% N2). SS1(ΔhpaA) was cultured in the same way as SS1 throughout the experiment, with the exception of the cultures being supplemented with 25 μg/ml kanamycin.

Growth curves.
SS1 and SS1(ΔhpaA) were first grown on Colombia-Iso plates to confluence for 2 to 3 days and then resuspended in 2 ml Brucella broth (Difco Laboratories) to an optical density at 600 nm (OD600) of 0.3 (1.5 × 109 bacteria/ml). Sixteen female C57BL/6 mice were orally infected with approximately 109 CFU of H. pylori SS1 or SS1(ΔhpaA) in Brucella broth under anesthesia (Isoflurane; Abbott Scandinavia Ab, Solna, Sweden) as previously described (27).

Detection of H. pylori SS1 (wild type) and SS1(ΔhpaA) in infected mice. (i) Quantitative culture.
The kinetics of SS1 in the colonization of mice have been well characterized, showing stable colonization between 2 and 8 weeks of infection (27). To determine the kinetics of colonization by SS1(ΔhpaA) in mice, animals were killed at various time points after infection (3 days, 3 weeks, and 8 weeks). The stomachs were removed and washed with phosphate-buffered saline to remove food residues. One half of the stomach was used for quantitative culture as previously described (27), and the other half was used for detection of H. pylori-specific genes by PCR. The stomach homogenates from the SS1(ΔhpaA)-infected mice were cultured on blood Skirrow plates both with and without kanamycin to examine if they had lost their antibiotic resistance during the gastric infection.

RESULTS
Comparison of the major proteome components in H. pylori strains SS1 and SS1(ΔhpaA).
To identify that no specific protein expression change had followed the construction of the HpaA mutant, we analyzed the proteome of H. pylori strain SS1 and its isogenic mutant by the 2-DE-based DIGE system. By use of cell lysis buffer compatible with the DIGE technology and isoelectric focusing at a pH interval of 3 to 10, over 800 distinct protein spots from each sample in the four replicates were detected by the DeCyder software and subsequent manual correction. The analysis of the expression profiles in strain SS1 and the SS1(ΔhpaA) mutant resulted in the identification of a minor number of spots (13) with a significantly changed level (P < 0.05). Of these spots, eight were found to be down-regulated and five spots were found to be upregulated in the SS1(ΔhpaA) mutant (Fig. ​(Fig.1).1). For identification of proteins, one preparative gel was stained with Sypro ruby, and spots were digested in gel and analyzed by nano-LC FT-ICR MS and MS/MS. We successfully identified the proteins shown in Table ​Table1.1. Notably, the trigger factor encoded by the tig gene located downstream of hpaA showed similar levels of expression in both strains (Fig. ​(Fig.11 and ​and2).2). However, Omp18 (HP0796) was detected in neither the wild-type strain nor the mutant. Thus, to ascertain that the disruption of the hpaA gene had not affected the transcription of its downstream gene, omp18, an omp18-specific RT-PCR was performed on SS1 and the SS1(ΔhpaA) mutant strain, which showed that Omp18 was transcribed in both strains (data not shown).

Detection of bacteria in infected mice.
Colonization of H. pylori was detected both by quantitative culture and by H. pylori-specific PCR. To evaluate the colonization pattern for SS1(ΔhpaA), mice were infected with either SS1(ΔhpaA) or SS1 as a reference and then killed at various time points ranging from 3 days to 2 months. Mice infected with SS1 showed a massive colonization at all time points studied, but bacteria could not be detected in the stomachs of mice infected with SS1(ΔhpaA) either by culture (Fig. ​(Fig.5)5) or by H. pylori-specific PCR at any time point (data not shown). To ascertain that SS1(ΔhpaA) had not lost its kanamycin resistance during the colonization in the stomach, the bacteria were grown on plates with and without kanamycin. However, no bacteria could be detected after culture on plates without kanamycin either (data not shown).

DISCUSSION
Many colonization and virulence factors have been evaluated as protective antigens in immunization studies in animal models (17, 22). For a bacterial protein to be considered as a candidate vaccine antigen, it should preferably be conserved (i.e., present in all strains), secreted or surface localized, and immunogenic (i.e., capable of stimulating the immune system). HpaA fulfills all these criteria; the gene encoding HpaA is present in and expressed by all H. pylori isolates (9, 39), indicating that it is valuable for the bacterium. Furthermore, H. pylori-infected subjects mount serum antibody responses against HpaA, which decline after eradication of the bacterium (23, 37), and HpaA induces maturation and antigen presentation of dendritic cells, showing its immunogenicity (36). In addition, it has been shown that HpaA is expressed both intracellularly and on the bacterial surface (20, 25).

To investigate the importance of HpaA in H. pylori infection, a previously described mutation of HpaA (25) was introduced into the mouse-adapted strain SS1, and the mutant strain was tested for its colonization ability and immunogenicity in a well-established animal model.

In order to verify that the mutation had not caused any damage on downstream genes or second-site mutations, we performed 2-D DIGE analysis to examine the overall protein expression pattern of H. pylori strain SS1. All the detected protein spots in the wild-type strain, with the exception of HpaA, were found in the mutant strain. However, 13 spots corresponding to 11 unique proteins showed small changes in expression levels in the mutant compared to the wild-type strain; of these, seven proteins were found to be down-regulated and four proteins were up-regulated. These identified proteins do not seem to be related on either the genetic or the functional level. In addition, it has been shown that minor changes in the protein expression level normally occur within a bacterial strain (35) (E. Carlsohn et al., unpublished data). The most important finding in the DIGE analysis of the wild type and its isogenic mutant was that the trigger factor encoded by the tig gene located downstream of hpaA showed similar levels of expression in both strains.

It is well known that OMPs tend to be discriminated in standard 2-DE displaying total cell extract. This is due both to poor solubility and low expression levels of the proteins of interest, and it is therefore important to design an appropriate isolation procedure for this protein species. We performed subcellular fractionation of OMPs in combination with one-dimensional PAGE analysis and nano-LC FT-ICR MS and MS/MS analyses of tryptic peptides. By use of this novel approach, we identified over 20 outer membrane proteins and 8 flagella-associated proteins in both investigated strains. All OMPs present in the wild-type strain, with the exception of HpaA, were also expressed in the mutant strain. The cotranscription of hpaA and the downstream gene omp18 has previously been described (20). It was therefore of interest to study the expression of the omp18 gene product in the constructed HpaA mutant to investigate possible polar effects on surrounding genes in the mutant. Unfortunately, the Omp18 protein was not detected in any of the strains. However, RT-PCR analysis of omp18 mRNA from the wild-type and mutant strains clearly showed that omp18 was transcribed in both strains, indicating that disruption of hpaA did not have any polar effects on its downstream genes (data not shown). In addition, to the best of our knowledge, the Omp18 protein has never been detected, suggesting that it might not be translated but that it might only be present on the mRNA level. Because no major differences between the two strains could be detected, we proceeded to an animal model for evaluation of the physiological importance of HpaA.

In vivo studies showed that while mice infected with the wild-type SS1 strain were heavily colonized, its isogenic mutant failed to colonize the mice at all time points examined. Thus, the fact that the mutant did not show significant differences in growth under laboratory conditions suggests that the observed phenotype is strictly in vivo dependent.

HpaA was originally pointed out as a putative N-acetylneuraminyllactose-binding hemagglutinin, and several studies have tried to elucidate the function of HpaA in in vitro adhesion studies, but the results are not conclusive. For example, bacterial binding to gastric cell lines in vitro was not affected by an inactivated hpaA gene (25). However, epithelial cell lines have been demonstrated to respond quite differently to bacterial stimulations compared to freshly isolated epithelial cells (4). Furthermore, deletion of the hpaA gene did not influence the glycosphingolipid recognition pattern of the bacteria, as evaluated by binding of the bacteria to previously identified H. pylori-binding glycosphingolipids on thin-layer chromatograms (1). Thus, both the parent SS1 strain and the HpaA knockout mutant bound to lactosylceramide, gangliotetraosylceramide, lactotetraosylceramide, and Leb-terminated glycosphingolipids (S. Teneberg et al., unpublished data). One may therefore speculate whether HpaA itself directly mediates receptor binding or whether it is involved in facilitating the adhesin transport and folding, or if it exerts regulatory functions. The role of HpaA needs to be elucidated in further investigations.

In conclusion, we have shown that the disruption of the HpaA-encoding gene did not induce any major differences in the protein expression pattern in the mutant compared with the wild-type strain. We have also demonstrated that HpaA is essential for bacterial colonization in the gastric mucosa of mice, establishing for the first time a physiological role of HpaA in vivo.

Abstract
Infection with the human gastric pathogen Helicobacter pylori can give rise to chronic gastritis, peptic ulcer, and gastric cancer. All H. pylori strains express the surface-localized protein HpaA, a promising candidate for a vaccine against H. pylori infection. To study the physiological importance of HpaA, a mutation of the hpaA gene was introduced into a mouse-adapted H. pylori strain. To justify that the interruption of the hpaA gene did not cause any polar effects of downstream genes or was associated with a second site mutation, the protein expression patterns of the mutant and wild-type strains were characterized by two different proteomic approaches. Two-dimensional differential in-gel electrophoresis analysis of whole-cell extracts and subcellular fractionation combined with nano-liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry for outer membrane protein profiling revealed only minor differences in the protein profile between the mutant and the wild-type strains. Therefore, the mutant strain was tested for its colonizing ability in a well-established mouse model. While inoculation with the wild-type strain resulted in heavily H. pylori-infected mice, the HpaA mutant strain was not able to establish colonization. Thus, by combining proteomic analysis and in vivo studies, we conclude that HpaA is essential for the colonization of H.