The use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid bacterial identification in patients with smear-positive bacterial meningitis.

OBJECTIVES: To assess the potential of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) in rapid identification of bacteria from smear-positive cerebrospinal fluid (CSF) in a cohort of patients with meningitis. METHODS: Single-centre observational study, including adults and children with community-acquired or postneurosurgical bacterial meningitis. Meningitis was defined using established criteria. Samples of CSF that had a positive CSF Gram stain were directly examined by MALDI-TOF-MS. Identification was considered accurate when identical to the CSF culture or PCR results (species and genus level). Laboratory workers performing the MALDI-TOF-MS and interpreting its results were blinded to the direct smear results, except for the fact that it was positive. MALDI-TOF-MS results were not conveyed to clinicians. RESULTS: MALDI-TOF-MS was tested on 44 CSF samples; ten samples were obtained from patients with community-acquired meningitis, and 34 samples were from patients with postneurosurgical meningitis. The assay identified bacteria correctly in 17/21 of the samples with Gram-negative rods observed on the direct smear, all obtained from patients who had undergone neurosurgery, (sensitivity 81%, 95% CI 64.2%-97.7%). In the postneurosurgical group, Gram-positive cocci were identified correctly in only 1/11 (9.1%) of the samples, and Candida species were not identified in two samples. Among patients with community-acquired meningitis, the assay did not identify Streptococcus pneumoniae in eight of eight samples, Neisseria meningitidis in one sample (1/1), and Streptococcus agalactiae in one sample (1/1). CONCLUSIONS: We found MALDI-TOF-MS to be useful in the rapid identification of Gram-negative rods directly from smear-positive CSF samples, but not of Gram-positive bacteria.

Pneumococcal IgA1 protease subverts specific protection by human IgA1.

Bacterial immunoglobulin A1 (IgA1) proteases may sabotage the protective effects of IgA. In vitro, both exogenous and endogenously produced IgA1 protease inhibited phagocytic killing of Streptococcus pneumoniae by capsule-specific IgA1 human monoclonal antibodies (hMAbs) but not IgA2. These IgA1 proteases cleaved and reduced binding of the the effector Fcα1 heavy chain but not the antigen-binding F(ab)/light chain to pneumococcal surfaces. In vivo, IgA1 protease-resistant IgA2, but not IgA1 protease-sensitive IgA1, supported 60% survival in mice infected with wild-type S. pneumoniae. IgA1 hMAbs protected mice against IgA1 protease-deficient but not -producing pneumococci. Parallel mouse sera with human IgA2 showed more efficient complement-mediated reductions in pneumococci with neutrophils than did IgA1, particularly with protease-producing organisms. After natural human pneumococcal bacteremia, purified serum IgG inhibited IgA1 protease activity in 7 of 11 patients (64%). These observations provide the first evidence in vivo that IgA1 protease can circumvent killing of S. pneumoniae by human IgA. Acquisition of IgA1 protease-neutralizing IgG after infection directs attention to IgA1 protease both as a determinant of successful colonization and infection and as a potential vaccine candidate.

Human milk lactoferrin is a serine protease that cleaves Haemophilus surface proteins at arginine-rich sites.

Lactoferrin is a member of the lactotransferrin family of non-haem, iron-binding glycoproteins and is found at high concentrations in all human secretions, where it plays a major role in mucosal defence. In recent work, we observed that lactoferrin has proteolytic activity and attenuates the pathogenic potential of Haemophilus influenzae by cleaving and removing two putative colonization factors, namely the IgA1 protease protein and the Hap adhesin. Experiments with protease inhibitors further suggested that lactoferrin may belong to a serine protease family. In the present study we explored the mechanism of lactoferrin protease activity and discovered that mutation of either Ser259 or Lys73 results in a dramatic decrease in proteolysis. Examination of the crystal structure revealed that these two residues are located in the N-terminal lobe of the protein, adjacent to a 12-15 A cleft that separates the N-lobe and the C-lobe and that can readily accommodate large polypeptide substrates. In additional work, we found that lactoferrin cleaves IgA1 protease at an arginine-rich region defined by amino acids 1379-1386 (RRSRRSVR) and digests Hap at an arginine-rich sequence between amino acids 1016 and 1023 (VRSRRAAR). Based on our results, we conclude that lactoferrin is a serine protease capable of cleaving arginine-rich sequences. We speculate that Ser259 and Lys73 form a catalytic dyad, reminiscent of a number of bacterial serine proteases. In addition, we speculate that lactoferrin may cleave arginine-rich sequences in a variety of microbial virulence proteins, contributing to its long-recognized antimicrobial properties.

Differential gene expression from two transcriptional units in the cag pathogenicity island of Helicobacter pylori.

Infection with Helicobacter pylori strains containing the cag Pathogenicity Island (cag PAI) is strongly correlated with the development of severe gastric disease, including gastric and duodenal ulceration, mucosa-associated lymphoid tissue lymphoma, and gastric carcinoma. Although in vitro studies have demonstrated that the expression of genes within the cag PAI leads to the activation of a strong host inflammatory response, the functions of most cag gene products and how they work in concert to promote an immunological response are unknown. We developed a transcriptional reporter that utilizes urease activity and in which nine putative regulatory sequences from the cag PAI were fused to the H. pylori ureB gene. These fusions were introduced in single copies onto the H. pylori chromosome without disruption of the cag PAI. Our analysis indicated that while each regulatory region confers a reproducible amount of promoter activity under laboratory conditions, they differ widely in levels of expression. Transcription initiating upstream of cag15 and upstream of cag21 is induced when the respective fusion strains are cocultured with an epithelial cell monolayer. Results of mouse colonization experiments with an H. pylori strain carrying the cag15-ureB fusion suggested that this putative regulatory region appears to be induced in vivo, demonstrating the importance of the urease reporter as a significant development toward identifying in vivo-induced gene expression in H. pylori.

Clinical pathology of foodborne diseases: notes on the patient with foodborne gastrointestinal illness.

The symptoms and signs in persons with food- or waterborne infections provide clues to the nature of the infecting microbe. Proper treatment of the affected individual, and protection of those exposed to the same source, is dependent on time-honored methods of diagnosis: exposure history, and physical examination. Laboratory testing may help to identify the responsible agent. Spontaneous recovery is the most likely outcome once supportive measures such as fluid and electrolyte replacement are addressed. Antibiotics are often unnecessary and may prolong fecal excretion of certain microorganisms. In immunosuppressed persons or those weakened by marginal nutrition, foodborne infection can be more severe, mandating more specific therapy. Management requires knowing the level of tissue invasion and organ infected by each of the commonly encountered microbes. Some of the most life-threatening infections (cholera, for example) are associated with no visible tissue injury, yet they have a profound impact on gut function. In contrast, salmonellosis and shigellosis can cause severe gut injury, and when foodborne infections extend beyond the confines of the gut, skilled care is essential. Examples are hemolytic uremic syndrome of Escherichia coli infections, or listeriosis, both of which require urgent attention. Long-term consequences of gut infections such as the paralytic Guillain-Barre syndrome following Campylobacter infections illustrates the long-term problems sometimes encountered. Because it is unlikely that all infectious agents will ever be removed from food and water in any country, sound medical intervention tailored to the extent of illness will be the mainstay of handling such illnesses.

Human lactoferrin proteolytic activity: analysis of the cleaved region in the IgA protease of Haemophilus influenzae.

Human lactoferrin proteolytically cleaves and inactivates two colonization factors of non-typable Haemophilus influenzae, the IgA protease precursor protein (Iga), and Hap, the non-pilus adhesin by which microoganisms adhere to host epithelial cells and form microcolonies. Iga and Hap are homologous proteins that are members of the autotransporter family of secreted proteins expressed by gram-negative bacteria. Studies of Iga cleaved by lactoferrin, reported here, show that proteolysis occurred within the helper region of Iga (Iga(beta)) domain which anchors the autotransporter within the Haemophilus outer membrane. The amino-terminus of the extracted Iga protein was not modified. The location of the proteolytic active site in human lactoferrin is under study. Lactoferrin proteolysis may attenuate pathogenicity of H. influenzae, an important cause of otitis media.

Role of Hpn and NixA of Helicobacter pylori in susceptibility and resistance to bismuth and other metal ions.

BACKGROUND: Helicobacter pylori produces Hpn, a 60-amino acid, histidine-rich protein that avidly binds nickel and zinc ions, and NixA, a high-affinity nickel transporter in the cytoplasmic membrane. We tested the hypothesis that Hpn and NixA govern susceptibility to metal ions in H. pylori. MATERIALS AND METHODS: Hpn-negative mutants of four H. pylori strains were constructed by standard allelic exchange techniques to yield isogenic Hpn+/Hpn-deficient pairs. A metal concentration that inhibited growth by 50% (IC50) was calculated for Ni2+, Zn2+, Cu2+, and Co2+ by comparing OD600 of cultures in metal-supplemented and control media. RESULTS: Among all four pairs of isogenic strains, the tolerance for Ni2+ was reduced significantly (p <.001) in the Hpn mutants; the mean IC50 value for wild-type strains was 1.9 mM; for the mutant, it was 0.8 mM. In contrast, growth inhibition by Zn2+ was identical within the fours pairs, as was Cu2+ and Co2+ tolerance in one pair tested. We also found that deletion of the hpn gene increases susceptibility to therapeutic forms of bismuth by testing a mutant and wild-type pair with ranitidine bismuth citrate, bismuth citrate, and four antibiotics. Minimal inhibitory concentrations of ranitidine bismuth citrate dropped from 9.2 to 2.3 microg/ml, and those of bismuth citrate dropped from 7.4 to 3.2 microg/ml (p <.05 for both comparisons), while susceptibility to the antibiotics was unaffected. Disruption of the nixA gene encoding the specific Ni2+ transport protein of H. pylori did not change susceptibility to bismuth. CONCLUSION: We concluded that bacteria lacking Hpn, cultured in vitro, are more susceptible than is the wild type to bismuth and Ni2+.

Human milk lactoferrin inactivates two putative colonization factors expressed by Haemophilus influenzae.

Haemophilus influenzae is a major cause of otitis media and other respiratory tract disease in children. The pathogenesis of disease begins with colonization of the upper respiratory mucosa, a process that involves evasion of local immune mechanisms and adherence to epithelial cells. Several studies have demonstrated that human milk is protective against H. influenzae colonization and disease. In the present study, we examined the effect of human milk on the H. influenzae IgA1 protease and Hap adhesin, two autotransported proteins that are presumed to facilitate colonization. Our results demonstrated that human milk lactoferrin efficiently extracted the IgA1 protease preprotein from the bacterial outer membrane. In addition, lactoferrin specifically degraded the Hap adhesin and abolished Hap-mediated adherence. Extraction of IgA1 protease and degradation of Hap were localized to the N-lobe of the bilobed lactoferrin molecule and were inhibited by serine protease inhibitors, suggesting that the lactoferrin N-lobe may contain serine protease activity. Additional experiments revealed no effect of lactoferrin on the H. influenzae P2, P5, and P6 outer-membrane proteins, which are distinguished from IgA1 protease and Hap by the lack of an N-terminal passenger domain or an extracellular linker region. These results suggest that human milk lactoferrin may attenuate the pathogenic potential of H. influenzae by selectively inactivating IgA1 protease and Hap, thereby interfering with colonization. Future studies should examine the therapeutic potential of lactoferrin, perhaps as a supplement in infant formulas.

Identification, cloning, and sequencing of the immunoglobulin A1 protease gene of Streptococcus pneumoniae.

The pneumococcus expresses a protease that hydrolyzes human immunoglobulin A1 (IgA1). A gene for IgA1 protease was identified from a plasmid library of pneumococcal DNA because of the effect of its overexpression on the colony morphology of Streptococcus pneumoniae. The deduced 1,964-amino-acid sequence is highly homologous to that of the IgA1 protease from Streptococcus sanguis. The similarity to the S. sanguis enzyme and the presence of a putative zinc-binding site suggest that the pneumococcal enzyme is a metalloprotease. The two streptococcal sequences differ in a hydrophilic region with 10 tandem repeats of a 20-mer in S. sanguis, which is replaced by a similar but less repetitive sequence in S. pneumoniae. Antiserum reactive with the pneumococcal IgA1 protease was used to demonstrate that the majority of the protein is cell associated. The expression and function of this gene were confirmed by insertional mutagenesis. Interruption of the chromosomal gene resulted in loss of expression of an approximately 200-kDa protein and complete elimination of detectable IgA1 protease activity.

Hydrogen uptake hydrogenase in Helicobacter pylori.

The peptic ulcer-causing bacterium Helicobacter pylori was found to contain an H2-uptake hydrogenase activity coupled to whole cell (aerobic) respiration. The activity was localized to membranes which functioned in the H2-oxidizing direction with a variety of artificial and physiological electron acceptors of positive redox potential. Immunoblotting of H. pylori membrane components with anti (B. japonicum) hydrogenase large and small subunit-specific antisera identified H. pylori hydrogenase peptides of approximately 65 and 26 kDa respectively, and H. pylori genomic DNA fragments hybridizing to the (B. japonicum) hydrogenase structural genes were identified. The membrane-bound activity was subject to anaerobic activation, like many NiFe hydrogenases. Difference absorption spectral studies revealed absorption peaks characteristic of b and c-type cytochromes, as well as of a bd-type terminal oxidase in the H. pylori H2-oxidizing membrane-associated respiratory chain.

Analysis of the specificity of bacterial immunoglobulin A (IgA) proteases by a comparative study of ape serum IgAs as substrates.

Immunoglobulin A (IgA) proteases are bacterial enzymes with substrate specificity for human serum and secretory IgAs. To further define the basis of this specificity, we examined the ability of IgA proteases of Clostridium ramosum, Streptococcus pneumoniae (EC 3.4.24.13), Neisseria meningitidis (EC 3.4.21.72), and Haemophilus influenzae (EC 3.4.21.72) to cleave serum IgAs of gorillas, chimpanzees, and orangutans. All enzymes cleaved the IgAs of the three apes despite differences in ape IgA1 hinge sequence relative to the human prototype. To directly compare the ape and human hinge cleavage sites, the sites were identified in eight ape IgA digests. This analysis confirmed that ape proteins were all cleaved in the IgA hinge region, in all but one case after proline residues. The exception, C. ramosum protease, cleaved gorilla and chimpanzee IgAs at peptide bonds having no proline, but the scissile bonds were in the same hinge location as the Pro-221-Val-222 cleaved in human IgA1. These data indicate that proline is not an invariant substrate requirement for all IgA proteases and that the location of the scissile bond, in addition to its composition, is a critical determinant of cleavage specificity.

Unique susceptibility of Helicobacter pylori to simethicone emulsifiers in alimentary therapeutic agents.

Helicobacter pylori is killed in vitro by polyoxyethylene acyl esters and ethers similar to simethicone emulsifiers in therapeutic antifoams. The MBC of these compounds for Helicobacter pylori was less than 20 micrograms/ml, while other gram-negative bacteria were unaffected by much higher concentrations of up to 50 mg/ml.

Lack of interference between IgA-binding proteins and IgA proteases of human pathogenic bacteria.

Some human bacterial pathogens produce specific immunoglobulin A1 (IgA1) proteases that cleave the heavy chain of human IgA1, generating intact Fab and Fc fragments. Other pathogenic bacterial species express surface proteins which bind to the Fc part of human IgA in a non-immune manner. To analyse whether IgA-binding proteins affect the activity of IgA1 proteases, the ability of seven different IgA1 proteases to hydrolyse IgA1 in the presence of either of two different bacterial IgA-binding proteins was tested. Data obtained in two different types of experiment suggest that IgA1 bound to IgA-binding proteins still functions as a substrate for IgA1 proteases. As Fc fragments produced by cleaving IgA1 with IgA1 proteases still bind to IgA-binding proteins, we conclude that these two types of bacterial protein act independently of each other.

Protein Hpn: cloning and characterization of a histidine-rich metal-binding polypeptide in Helicobacter pylori and Helicobacter mustelae.

Helicobacter pylori is a human gastrointestinal pathogen involved in gastritis, duodenal ulcers, and gastric neoplasia. This microorganism produces large amounts of a urease which, like all known ureases, has nickel in the active site. We have identified a protein in clinical isolates of H. pylori and an identical protein in the ferret pathogen Helicobacter mustelae that strongly binds Ni2+ and Zn2+. This protein has been named Hpn to emphasize its origins in H. pylori and its affinity for nickel. The encoding hpn gene, cloned and expressed in Escherichia coli ER1793, has an open reading frame (180 bp) that specifies a protein with a calculated molecular mass of 7,077 Da and with the same amino-terminal sequence as that of wild-type Hpn. The deduced sequence of Hpn consists of 60 amino acids, of which 28 (47%) are histidines. The hpn gene does not map with the urease gene cluster on the H. pylori chromosome. An Hpn-negative, isogenic H. pylori strain, generated by hpn gene deletion and grown on blood agar, had the same urease activity that wild-type cells did. Thus, the role of Hpn in helicobacters is unknown.

Post-infectious human serum antibodies inhibit IgA1 proteinases by interaction with the cleavage site specificity determinant.

Bacterial pathogens of the genera Neisseria and Haemophilus secrete IgA1 proteinases which cleave human IgA1 in the heavy chain hinge region. The exact peptide bond cleaved is strain-dependent, but remains invariant despite repeated subculture. Haemophilus influenzae and Neisseria meningitidis produce proteinases of two cleavage site specificities (type 1 and type 2). We examined serial acute and convalescent sera from patients recovering from meningitis due to N. meningitidis or H. influenzae, and found a significant rise in serum titer of inhibitory antibodies against these enzymes. In each case the proteinase from the infecting organism was more susceptible to inhibition than were proteinases from that genus that had different cleavage specificity. Inhibition of sixteen type 1-type 2 hybrid H. influenzae IgA1 proteinases revealed complete concordance between inhibitory titer and cleavage site specificity. Inhibition of hybrid proteinases differing in a 123 amino acid segment known to determine cleavage site specificity (termed the CSD) further localized the site of antibody action to this site. These results from a limited number of patients with natural infections suggest that inhibiting antibody recognizes epitopes within the CSD. Alternatively, antibody may bind to epitopes outside the CSD and inhibit via steric hindrance.