Serum-sensitive Strains Research Articles

Comparison of lipooligosaccharides from human challenge strains of Neisseria gonorrhoeae.

The alarming rise of antibiotic resistance and the emergence of new vaccine technologies have increased the focus on vaccination to control gonorrhea. Neisseria gonorrhoeae strains FA1090 and MS11 have been used in challenge studies in human males. We used negative-ion MALDI-TOF MS to profile intact lipooligosaccharide (LOS) from strains MS11mkA, MS11mkC, FA1090 A23a, and FA1090 1-81-S2. The MS11mkC and 1-81-S2 variants were isolated from male volunteers infected with MS11mkA and A23a, respectively. LOS profiles were obtained after purification using the classical phenol water extraction method and by microwave-enhanced enzymatic digestion, which is more amenable for small-scale work. Despite detecting some differences in the LOS profiles, the same major species were observed, indicating that microwave-enhanced enzymatic digestion is appropriate for MS studies. The compositions determined for MS11mkA and mkC LOS were consistent with previous reports. FA1090 is strongly recognized by mAb 2C7, an antibody-binding LOS with both α- and β-chains if the latter is a lactosyl group. The spectra of the A23a and 1-81-S2 FA1090 LOS were similar to each other and consistent with the expression of α-chain lacto-N-neotetraose and β-chain lactosyl moieties that can both be acceptor sites for sialic acid substitution. 1-81-S2 LOS was analyzed after culture with and without media supplemented with cytidine-5'-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac), which N. gonorrhoeae needs to sialylate its LOS. LOS sialylation reduces the infectivity of gonococci in men, although it induces serum resistance in serum-sensitive strains and reduces killing by neutrophils and antimicrobial peptides. The infectivity of FA1090 in men is much lower than that of MS11mkC, but the reason for this difference is unclear. Interestingly, some peaks in the spectra of 1-81-S2 LOS after bacterial culture with CMP-Neu5Ac were consistent with disialylation of the LOS, which could be relevant to the reduced infectivity of FA1090 in men and could have implications regarding the phase variation of the LOS and the natural history of infection.

Functional characterization of the gonococcal polyphosphate pseudo-capsule.

Neisseria gonorrhoeae is an exclusively human pathogen able to evade the host immune system through multiple mechanisms. Gonococci accumulate a large portion of phosphate moieties as polyphosphate (polyP) on the exterior of the cell. Although its polyanionic nature has suggested that it may form a protective shield on the cell surface, its role remains controversial. Taking advantage of a recombinant His-tagged polyP-binding protein, the presence of a polyP pseudo-capsule in gonococcus was demonstrated. Interestingly, the polyP pseudo-capsule was found to be present in specific strains only. To investigate its putative role in host immune evasion mechanisms, such as resistance to serum bactericidal activity, antimicrobial peptides and phagocytosis, the enzymes involved in polyP metabolism were genetically deleted, generating mutants with altered polyP external content. The mutants with lower polyP content on their surface compared to the wild-type strains, became sensitive to complement-mediated killing in presence of normal human serum. Conversely, naturally serum sensitive strains that did not display a significant polyP pseudo-capsule became resistant to complement in the presence of exogenous polyP. The presence of polyP pseudo-capsule was also critical in the protection from antibacterial activity of cationic antimicrobial peptide, such as cathelicidin LL-37. Results showed that the minimum bactericidal concentration was lower in strains lacking polyP than in those harboring the pseudo-capsule. Data referring to phagocytic killing resistance, assessed by using neutrophil-like cells, showed a significant decrease in viability of mutants lacking polyP on their cell surface in comparison to the wild-type strain. The addition of exogenous polyP overturned the killing phenotype of sensitive strains suggesting that gonococcus could exploit environmental polyP to survive to complement-mediated, cathelicidin and intracellular killing. Taken together, data presented here indicate an essential role of the polyP pseudo-capsule in the gonococcal pathogenesis, opening new perspective on gonococcal biology and more effective treatments.

Yersinia pestis Δail Mutants Are Not Susceptible to Human Complement Bactericidal Activity in the Flea.

Ail confers serum resistance in humans and is a critical virulence factor of Y. pestis, the causative agent of plague. Here, the contribution of Ail for Y. pestis survival in the flea vector was examined. Rat or human but not mouse sera were bactericidal against a Y. pestis Δail mutant at 28°C in vitro. Complement components deposited rapidly on the Y. pestis surface as measured by immunofluorescent microscopy. Ail reduced the amount of active C3b on the Y. pestis surface. Human sera retained bactericidal activity against a Y. pestis Δail mutant in the presence of mouse sera. However, in the flea vector, the serum protective properties of Ail were not required. Flea colonization studies using murine sera and Y. pestis KIM6+ wild type, a Δail mutant, and the Δail/ail+ control showed no differences in bacterial prevalence or numbers during the early stage of flea colonization. Similarly, flea studies with human blood showed Ail was not required for serum resistance. Finally, a variant of Ail (AilF100V E108_S109insS) from a human serum-sensitive Y. pestis subsp. microtus bv. Caucasica 1146 conferred resistance to human complement when expressed in the Y. pestis KIM6+ Δail mutant. This indicated that Ail activity was somehow blocked, most likely by lipooligosaccharide, in this serum sensitive strain. IMPORTANCE This work contributes to our understanding of how highly virulent Y. pestis evolved from its innocuous enteric predecessor. Among identified virulence factors is the attachment invasion locus protein, Ail, that is required to protect Y. pestis from serum complement in all mammals tested except mice. Murine sera is not bactericidal. In this study, we asked, is bactericidal sera from humans active in Y. pestis colonized fleas? We found it was not. The importance of this observation is that it identifies a protective niche for the growth of serum sensitive and nonsensitive Y. pestis strains.

Outer membrane vesicle-mediated serum protection in Aggregatibacter actinomycetemcomitans

ABSTRACT Aggregatibacter actinomycetemcomitans belongs to the HACEK group of fastidious Gram-negative organisms, a recognized cause of infective endocarditis. A. actinomycetemcomitans is also implicated in periodontitis, with rapid progress in adolescents. We recently demonstrated that the major outer membrane protein, OmpA1 was critical for serum survival of the A. actinomycetemcomitans serotype a model strain, D7SS, and that the paralogue, OmpA2 could operate as a functional homologue to OmpA1 in mediating serum resistance. In the present work, an essentially serum-sensitive ompA1 ompA2 double mutant A. actinomycetemcomitans strain derivative was exploited to elucidate if A. actinomycetemcomitans OMVs can contribute to bacterial serum resistance. Indeed, supplementation of OMVs resulted in a dose-dependent increase of the survival of the serum-sensitive strain in incubations in 50% normal human serum (NHS). Whereas neither OmpA1 nor OmpA2 was required for the OMV-mediated serum protection, OMVs and LPS from an A. actinomycetemcomitans strain lacking the LPS O-antigen polysaccharide part were significantly impaired in protecting D7SS ompA1 ompA2. Our results using a complement system screen assay support a model where A. actinomycetemcomitans OMVs can act as a decoy, which can trigger complement activation in an LPS-dependent manner, and consume complement components to protect serum-susceptible bacterial cells.

Expression of Rickettsia Adr2 protein in E. coli is sufficient to promote resistance to complement-mediated killing, but not adherence to mammalian cells.

Bacteria exposed to host serum are subject to the antibacterial effects to the complement system. However, pathogenic microorganisms have evolved mechanisms of evading this immune attack. We have previously demonstrated that at least two R. conorii antigens, RC1281/Adr1 and OmpB β-peptide, contribute to the evasion of complement-mediated killing by binding the complement regulatory proteins vitronectin and factor H. RC1282/Adr2, a protein related to Adr1, is predicted to share similar structural features, suggesting that this protein may also contribute to evasion of complement-mediated killing. Interestingly, the R. prowazekii Adr1 and Adr2(RP828) proteins were originally found to interact with host cell surface proteins, suggesting their putative roles as adhesins in this pathogenic rickettsial species. In this study, we expressed both R. conorii and R. prowazekii Adr2 on the surface of a non-adherent, serum-sensitive strain of E. coli to examine the potential role of this protein to mediate evasion of complement-mediated killing and adherence to host cells. We demonstrate that, similar to R. conorii Adr1, R. conorii and R. prowazekii Adr2 are sufficient to mediate serum resistance and to promote interaction with the host complement regulator vitronectin. Furthermore, we demonstrate that expression of Adr2 in a non-adherent strain of E. coli is insufficient to mediate adherence to cultured mammalian endothelial cells. Together, our data demonstrate that the R. conorii and R. prowazekii Adr2 protein does not participate in the interactions with mammalian cells, but rather, participates in the evasion of killing by complement.

Cronobacter sakazakii clinical isolates overcome host barriers and evade the immune response

Cronobacter sakazakii is the most frequently clinically isolated species of the Cronobacter genus. However the virulence factors of C. sakazakii including their ability to overcome host barriers remains poorly studied. In this study, ten clinical isolates of C. sakazakii were assessed for their ability to invade and translocate through human colonic carcinoma epithelial cells (Caco-2) and human brain microvascular endothelial cells (HBMEC). Their ability to avoid phagocytosis in human macrophages U937 and human brain microglial cells was investigated. Additionally, they were tested for serum sensitivity and the presence of the Cronobacter plasminogen activation gene (cpa) gene, which is reported to confer serum resistance.Our data showed that the clinical C. sakazakii strains invaded and translocated through Caco-2 and HBMEC cell lines and some strains showed significantly higher levels of invasion and translocation. Moreover, C. sakazakii was able to persist and even multiply in phagocytic macrophage and microglial cells. All strains, except one, were able to withstand human serum exposure, the single serum sensitive strain was also the only one which did not encode for the cpa gene. These results demonstrate that C. sakazakii clinical isolates are able to overcome host barriers and evade the host immune response indicating their capacity to cause diseases such as necrotizing enterocolitis (NEC) and meningitis. Our data showed for the first time the ability of C. sakazakii clinical isolates to survive and multiply within human microglial cells. Additionally, it was shown that C. sakazakii clinical strains have the capacity to translocate through the Caco-2 and HBMEC cell lines paracellularly.

α-2,3-sialyltransferase expression level impacts the kinetics of lipooligosaccharide sialylation, complement resistance, and the ability of Neisseria gonorrhoeae to colonize the murine genital tract.

ABSTRACT Neisseria meningitidis and Neisseria gonorrhoeae modify the terminal lacto-N-neotetraose moiety of their lipooligosaccharide (LOS) with sialic acid. N. gonorrhoeae LOS sialylation blocks killing by complement, which is mediated at least in part by enhanced binding of the complement inhibitor factor H (FH). The role of LOS sialylation in resistance of N. meningitidis to serum killing is less well defined. Sialylation in each species is catalyzed by the enzyme LOS α-2,3-sialyltransferase (Lst). Previous studies have shown increased Lst activity in N. gonorrhoeae compared to N. meningitidis due to an ~5-fold increase in lst transcription. Using isogenic N. gonorrhoeae strains engineered to express gonococcal lst from either the N. gonorrhoeae or N. meningitidis lst promoter, we show that decreased expression of lst (driven by the N. meningitidis promoter) reduced LOS sialylation as determined by less incorporation of tritium-labeled cytidine monophospho-N-acetylneuraminic acid (CMP-NANA; the donor molecule for sialic acid). Diminished LOS sialylation resulted in reduced rates of FH binding and increased pathway activation compared to N. gonorrhoeae promoter-driven lst expression. The N. meningitidis lst promoter generated sufficient Lst to sialylate N. gonorrhoeae LOS in vivo, and the level of sialylation after 24 h in the mouse genital tract was sufficient to mediate resistance to human serum ex vivo. Despite demonstrable LOS sialylation in vivo, gonococci harboring the N. meningitidis lst promoter were outcompeted by those with the N. gonorrhoeae lst promoter during coinfection of the vaginal tract of estradiol-treated mice. These data highlight the importance of high lst expression levels for gonococcal pathogenesis.

Construction of an iss deleted mutant strain from a native avian pathogenic Escherichia coli O78: K80 and in vitro serum resistance evaluation of mutant

BACKGROUND: Colibacillosis, caused by different serotypes of avian pathogenic Escherichia coli (APEC), is one of the important diseases in poultry industry. The isolate O78 is the most prevalent serotype of APEC in Iran. One of the APEC virulence factors, increased serum survival (iss) gene, is related to serum resistance. The usual form of colibacillosis in avian is extraintestinal, and serum resistance is applied one way by APEC to reach internal organs; hence, it appears that the control of colibacillosis in poultry regarding the deletion of iss and the construction of a serum sensitive APEC strain is beneficial. Additionally, the knowledge about APEC serum resistance could be extended using mutant strains. OBJECTIVES: The present study was an attempt to generate an iss mutant strain from native APEC-O78 strain ||and to study the level of serum resistance of native APEC-O78 strain χ1378 in comparison with its mutant (APEC-O78 strain χ1378||). METHODS: The lambda red recombinase system was utilized to delete iss gene in native APEC-O78 strain χ1378. This strain was first transformed with the plasmid pkD46 to introduce the lambda red recombinase system and then the PCR product with sequence homology to the iss gene and a kanamycin resistance marker was transformed into the APEC-O78 strain χ1378. Serum sensitivity of mutant and wild type strain was investigated by microtiter test. RESULTS: The generation of mutant was successful and the iss was replaced with kanamycin resistance cassette. Also, it was observed that the mutant was sensitive to serum. However, serum sensitivity of iss deleted mutant was not statistically different from its parents. CONCLUSIONS:Application of lambda red recombination could be a simple and useful technique for production of a precisely defined gene deletion. Also, there may be some genes that compensate the activity of iss gene.

Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge.

Natural IgG antibodies in normal rabbit serum are involved in killing of the ompP2 mutant of Haemophilus parasuis SC096 strain via the classical complement pathway

Serum resistance in Haemophilus parasuis strain SC096 has been shown to be dependent on expression of the outer membrane protein P2 (OmpP2) and loss of the ompP2 gene results in significantly greater sensitivity to both porcine and rabbit sera. However, the mechanism of complement activation by the serum sensitive ΔompP2 strain is unknown. In this study, the classical complement pathway is demonstrated to be the main pathway for killing the H. parasuis ΔompP2 strain, and not the mannan-binding lectin (MBL) or alternative pathway. In addition, absorption of antibodies against ΔompP2 strain or depletion of IgGs from serum inhibited serum killing activity, which could be restored by addition of heat-inactivated serum or purified IgGs. Western blot analysis indicated that the OmpP2 mutant could bind significantly more IgGs than the wild type strain SC096 when incubated with serum. Finally, IgGs in normal rabbit serum targeted to the OMPs, but not lipooligosaccharide (LOS) in the OmpP2 mutant strain were found to be involved in bacterial killing indicating that the bactericidal epitope(s) is in the outer membrane proteins.

Serum sensitivity of Pseudomonas aeruginosa isolated from sputum as a virulence factor in the lower respiratory tract

To elucidate the clinical significance of serum-sensitivity of respiratory pathogenic Pseudomonas aeruginosa (P. aeruginosa) strains, we examined serum-sensitivity of P. aeruginosa isolated from 16 patients with lower respiratory tract infections and clinical backgrounds of these patients. We also evaluated the virulence of four serum-resistant and four serum-sensitive P. aeruginosa strains in murine pneumonia model induced by intratracheal challenge, and the silver-stained profiles of purified lipopolysaccharide (LPS) from these strains. Serum-sensitive strains were isolated only from patients with chronic bronchitis, bronchiectasis, and diffuse panbronchiolitis colonized with P. aeruginosa, and rarely caused pneumonias, while serum-resistant strains caused pneumonias in some cases. Intratracheal challenge of mice with 5 x 10(7) cfu per mouse of a serum resistant strain caused fatal hemorrhagic pneumonia with bacteremia. In contrast, the same dose of a serum-sensitive strain provided non-fatal pneumonia without bacteremia. LD50 of serum-sensitive strains in a murine model of P. aeruginosa pneumonia were at least 2-10 times higher than those of serum-resistant strain. The LPS profiles of two serum-resistant strains and one serum-sensitive strain showed ladder-like patterns. The similar analysis demonstrated that one serum-sensitive strain was lack of ladder-like patterns. These data support that serum-sensitive P. aeruginosa strains are less virulent than serum-resistant P. aeruginosa strains in the lower respiratory tract, and serum sensitivity of P. aeruginosa strains is determined by the structure of the O-side chain of LPS; either lack of the O-side chain or the presence of sparse O-side chain.

Immune Evasion ofLeptospiraSpecies by Acquisition of Human Complement Regulator C4BP

Leptospirosis is a spirochetal zoonotic disease of global distribution with a high incidence in tropical regions. In the last 15 years it has been recognized as an important emerging infectious disease due to the occurrence of large outbreaks in warm-climate countries and, occasionally, in temperate regions. Pathogenic leptospires efficiently colonize target organs after penetrating the host. Their invasiveness is attributed to the ability to multiply in blood, adhere to host cells, and penetrate into tissues. Therefore, they must be able to evade the innate host defense. The main purpose of the present study was to evaluate how several Leptospira strains evade the protective function of the complement system. The serum resistance of six Leptospira strains was analyzed. We demonstrate that the pathogenic strain isolated from infected hamsters avoids serum bactericidal activity more efficiently than the culture-attenuated or the nonpathogenic Leptospira strains. Moreover, both the alternative and the classical pathways of complement seem to be responsible for the killing of leptospires. Serum-resistant and serum-intermediate strains are able to bind C4BP, whereas the serum-sensitive strain Patoc I is not. Surface-bound C4BP promotes factor I-mediated cleavage of C4b. Accordingly, we found that pathogenic strains displayed reduced deposition of the late complement components C5 to C9 upon exposure to serum. We conclude that binding of C4BP contributes to leptospiral serum resistance against host complement.

LgtC Expression Modulates Resistance to C4b Deposition on an Invasive Nontypeable Haemophilus influenzae

We have previously shown that C3 binding to serum-resistant nontypeable Haemophilus influenzae (NTHi) strain R2866 is slower than C3 binding to a serum-sensitive strain. Ab-dependent classical pathway activation is required for complement-dependent killing of NTHi. To further characterize the mechanism(s) of serum resistance of R2866, we compared binding of complement component C4b to R2866 with a serum-sensitive variant, R3392. We show that C4b binding to R2866 relative to R3392 was delayed, suggesting regulation of the classical pathway of complement. Increased C4b deposition on R3392 was independent of the amount and subclass of Ab binding, suggesting that an impediment to C4b binding existed on R2866. Immunoblotting and mass spectrometry indicated that lipooligosaccharide and outer membrane proteins P2 and P5 were targets for C4b. P2 and P5 sequences and expression levels were similar in both strains. Insertional inactivation of the phase-variable lipooligosaccharide biosynthesis gene lgtC in R2866 augmented C4b deposition to levels seen with R3392 and rendered the bacteria sensitive to serum and whole blood. These results suggest a direct role of lgtC expression in the inhibition of C4b deposition and consequent serum resistance of R2866. Alteration of surface glycans of NTHi may be a critical event in determining the ability of a strain to evade host defenses and cause disseminated infection.

Role of Complement in Defense of the Middle Ear Revealed by Restoring the Virulence of NontypeableHaemophilus influenzae siaBMutants

Nontypeable (NT) Haemophilus influenzae is an important cause of otitis media in children. We have shown previously that NT H. influenzae mutants defective in their ability to sialylate lipopolysaccharide (LPS), called siaB mutants, show attenuated virulence in a chinchilla model of experimental otitis media (EOM). We show that complement is a key arm of host innate immunity against NT H. influenzae-induced EOM. Depleting complement in chinchillas by use of cobra venom factor (CoVF) rendered two otherwise avirulent siaB mutants fully virulent and able to cause EOM with severity similar to that of wild-type strains. Clearance of infection caused by siaB mutants in CoVF-treated animals coincided with reappearance of C3. Wild-type strains were more resistant to direct complement-mediated killing than their siaB mutants. The serum-resistant strain bound less C3 and C4 than the serum-sensitive strain. Neither NT H. influenzae strain tested bound factor H (alternative complement pathway regulator). Selective activation of the alternative pathway resulted in more C3 binding to siaB mutants. LPS sialylation had a more profound impact on the amount of alternative-pathway-mediated C3 binding ( approximately 5-fold decrease in fluorescence) when LPS was the main C3 target, as occurred on the more serum-resistant strain. In contrast, only an approximately 1.5-fold decrease in fluorescence intensity of C3 binding was seen with the serum-sensitive strain, where surface proteins predominantly bound C3. Differences in binding sites for C3 and C4 may account for variations in serum resistance between NT H. influenzae strains, which in turn may impact their virulence. These data demonstrate a central role for complement in innate immune defenses against NT H. influenzae infections and specifically EOM.

Heterogeneity in Tandem Octanucleotides within Haemophilus influenzae Lipopolysaccharide Biosynthetic Gene losA Affects Serum Resistance

Haemophilus influenzae is subject to phase variation mediated by changes in the length of simple sequence repeat regions within several genes, most of which encode either surface proteins or enzymes involved in the synthesis of lipopolysaccharides (LPS). The translational repeat regions that have been described thus far all consist of tandemly repeated tetranucleotides. We describe an octanucleotide repeat region within a putative LPS biosynthetic gene, losA. Approximately 20 percent of nontypeable H. influenzae strains contain copies of losA and losB in a genetic locus flanked by infA and ksgA. Of 30 strains containing losA at this site, 24 contained 2 tandem copies of the octanucleotide CGAGCATA, allowing full-length translation of losA (on), and 6 strains contained 3, 4, 6, or 10 tandem copies (losA off). For a serum-sensitive strain, R3063, with losA off (10 repeat units), selection for serum-resistant variants yielded a heterogeneous population in which colonies with increased serum resistance had losA on (2, 8, or 11 repeat units), and colonies with unchanged sensitivity to serum had 10 repeats. Inactivation of losA in strains R3063 and R2846 (strain 12) by insertion of the cat gene decreased the serum resistance of these strains compared to losA-on variants and altered the electrophoretic mobility of LPS. We conclude that expression of losA, a gene that contributes to LPS structure and affects serum resistance, is determined by octanucleotide repeat variation.

Pseudomonas aeruginosa Strains from Nosocomial Pneumonia Are More Serum Resistant than P. aeruginosa Strains from Noninfectious Respiratory Colonization Processes

Serum resistance is regarded as a major virulence factor of bacteria and is thought to be mediated by O side chains of the lipopolysaccharides (LPS). We investigated the serum-resistance properties and O serogroups of Pseudomonas aeruginosa strains isolated from intensive careunit (ICU) patients with pneumonia and from the respiratory tract of ICU patients without respiratory tract infections. 171 P. aeruginosa strains were consecutively isolated from bronchoalveolar lavage fluid or transtracheal aspirates of ICU patients with monobacterial nosocomial pneumonia and 49 strains were isolated from the respiratory tract of ICU patients without respiratory tract infections. All strains were O serogrouped using Oantigen- specific sera for 14 O serogroups and tested for their sensitivity to the serum's bactericidal effect. Using two different analyses, the frequency of serum-sensitive isolates was significantly lower in strains from patients with pneumonia (56.1%; n = 96/171 and 22.8%, n = 39/171, respectively) than in strains from asymptomatically colonized patients (73.46%; 36/49 and 38.8%, n = 19/49, respectively) (p = 0.03; OR = 2.163; 95% CI = 1.072-4.368 and p = 0.0289; OR = 2.144; 95% CI = 1.089-4.368, respectively). O serogrouping revealed higher frequency of the serogroups A (11.9% and 16.3%, respectively), B (14.3% and 21%), E (26.5% and 24.6%), and I (28.6% and 28%) in both strain collections. The frequency of serum-sensitive strains (13/28 and 3/45, respectively) was significantly lower among strains expressing the A and B serogroups, than for all other serogroups (p < 0.05). Strains isolated from patients with pneumonia and strains possessing O-A or O-B serogroups appear to have greater pathogenic potential by virtue of their ability to resist serum-mediated killing. The linkage, however, between the O serogroups, serum resistance, and a strain's virulence remains unclear at this stage.

Regulation of complement activation at the C3-level by serum resistant leptospires

Leptospires are spirochetes that are transmitted to humans through contacts with wild or domestic animals or via an exposure to contaminated soil or water. In this study we have compared the serum-sensitivity of five pathogenic strains of leptospires ( L. interrogans) to an environmental isolate (strain Patoc). Different levels of sensitivities to human serum were seen. Interestingly, the most sensitive strain was the non-pathogenic Patoc strain. The fully and intermediately resistant strains have been isolated from human patients. Testing was performed in the absence of specific antibodies, and killing was found to be dependent on the complement system. The serum sensitive Patoc strain was killed in human serum within minutes, whereas the most resistant strains tolerated serum up to 4 h. We also tested the deposition of the complement components C3, C5, C6, C8 and C5b-9 to the surfaces of the sensitive and resistant strains of Leptospira by immunofluorescence microscopy and ELISA. C3 was deposited on both the sensitive and resistant strains, but the terminal complement components were detected only on the surface of the complement-sensitive strain. The complement resistant and intermediate strains were found to bind more factor H from human serum than the complement sensitive strain. Thus, binding of this major alternative complement pathway inhibitor is related to serum resistance in Leptospira spirochetes.

A C1q-binding 40 kDa porin from Aeromonas salmonicida: Cloning, sequencing, role in serum susceptibility and fish immunoprotection

A 40 kDa C1q-binding outer membrane protein from Aeromonas salmonicida was identified by direct-binding assay with biotinylated C1q, a subcomponent of the complement classical pathway component C1. The 40 kDa porin structural gene from the A450 A. salmonicida typical strain (A +:O +) was cloned in Escherichia coli and sequenced. The amino acid sequence of the 40 kDa A. salmonicida porin, its ability to bind C1q in an antibody independent process, and its immunological cross-reaction with the A. hydrophila AH-3 porin II, allow us to determine the role of this protein in serum susceptibility. Furthermore, we obtained defined A. salmonicida 40 kDa porin insertion mutants in serum sensitive or resistant strains, and we complemented these mutants with a plasmid harboring only the 40 kDa porin gene from A. salmonicida A450 in order to define its role as an important surface molecule involved in serum susceptibility and C1q binding. Similar complementation was obtained using the A. hydrophila AH-3 porin II gene. The 40 kDa porin gene and/or protein was present in all the A. salmonicida typical or atypical strains tested. Furthermore, the A. hydrophila AH-3 porin II seems to be an important molecule for fish immunoprotection against either A. salmonicida or A. hydrophila strains.

Normal Human Sera Contain Bactericidal IgG That Binds to the Oligosaccharide Epitope Expressed within Lipooligosaccharides of Neisseria gonorrhoeae

Although more than several investigators reported the presence of antibodies in normal human sera (NHS) that bind to lipooligosaccharide (LOS) of Neisseria gonorrhoeae, the specificities of those antibodies were not fully characterized. To identify anti-LOS antibodies in NHS, we used LOS from a serum-sensitive strain, JW31R, as an affinity ligand and purified IgG from NHS that bound to JW31R LOS. The affinity purified IgG (AP-IgG) binds to the oligosaccharide (OS) moiety of both the ligand LOS and its truncated form, 15253 LOS. Lipid A could be essential for maximum expression of the carbohydrate epitope that resides on 15253 OS. We also found that AP-IgG is capable of killing a serum-sensitive strain JW31R. The present work provided direct evidence that NHS contain bactericidal antibodies specific for a site close to the inner core OS expressed on gonococcal LOS. The present results not only show that anti-LOS antibodies specific for the inner core OS could play a major role in our defense against gram-negative bacteria. But also they demonstrated that such core OS or a nearby site could be utilized as possible targets for vaccine development against microbial infections.

The UspA2 Protein of Moraxella catarrhalis Is Directly Involved in the Expression of Serum Resistance

Many strains of Moraxella catarrhalis are resistant to the bactericidal activity of normal human serum. Previous studies have shown that mutations involving the insertion of an antibiotic resistance cartridge into the M. catarrhalis uspA2 gene resulted in the conversion of a serum-resistant strain to a serum-sensitive phenotype. In the present study, the deletion of the entire uspA2 gene from the serum-resistant M. catarrhalis strain O35E resulted in a serum-sensitive phenotype and did not affect either the rate of growth or the lipooligosaccharide expression profile of this mutant. Inactivation of the classical complement pathway in normal human serum with Mg2+ and EGTA resulted in the survival of this uspA2 mutant. In contrast, blocking of the alternative complement pathway did not protect this uspA2 mutant from complement-mediated killing. To determine whether the UspA2 protein is directly involved in serum resistance, transformation and allelic exchange were used to replace the uspA2 gene in the serum-resistant strain O35E with the uspA2 gene from the serum-sensitive M. catarrhalis strain MC317. The resultant O35E transformant exhibited a serum-sensitive phenotype. Similarly, when the uspA2 gene from the serum-resistant strain O35E was used to replace the uspA2 gene in the serum-sensitive strain MC317, the MC317 transformant acquired serum resistance. The use of hybrid O35E-MC317 uspA2 genes showed that the N-terminal half of the O35E protein contained a 102-amino-acid region that was involved in the expression of serum resistance. In addition, when the uspA2 genes from strains O35E and MC317 were cloned and expressed in Haemophilus influenzae DB117, only the O35E UspA2 protein caused a significant increase in the serum resistance of the H. influenzae recombinant strain. These results prove that the UspA2 protein is directly involved in the expression of serum resistance by certain M. catarrhalis strains.