Interaction of clinical isolates of nonencapsulated Haemophilus influenzae with mammalian extracellular matrix proteins

The adherence of clinical isolates of nonencapsulated Haemophilus influenzae strains from patients with chronic bronchitis to distinct immobilized extracellular matrix components was determined. With selected strains the induction of plasmin formation by these isolates was studied. The strains could be divided into two groups: strains that showed a very high level of adherence to laminin and type I collagen, as well as adhesion to fibronectin and strains that showed only a moderate level of adhesion to laminin and a low level of adhesion to fibronectin. Plasmin formation was demonstrated for three out of eight isolates. Persisting and nonpersisting strains did not differ quantitatively or qualitatively with respect to the level of adhesiveness to the distinct matrix proteins and in their ability to induce plasmin formation.     

流感嗜血杆菌的基因组学研究进展

流感嗜血杆菌Rd型是第一个被测序的细菌.其基因组大小为1830137bp,A含量为31%,C为19%,G为19%,T为31%,GC含量为38%,TIGR确认了1473个具有重要功能的基因.基因组序列中包含有复制子、核糖体启动子、毒力基因、DNA转运系统、调节子等结构.流感嗜血杆菌的基因组研究必将对寻找疾病相关基因和抗原基因,筛选药物作用靶位,研制特异的诊断试剂、药物和疫苗具有十分重要的科学意义.对流感嗜血杆菌的基因组学研究进展进行了综述.     

流感嗜血杆菌的分型研究

目的分析同济医院分离的流感嗜血杆菌的生物学分型及荚膜基因分型,了解本地区分离的流感嗜血杆菌的主要流行株。方法2012年1月1日至2012年12月31日从华中科技大学同济医学院附属同济医院分离流感嗜血杆菌100株。根据脲酶、吲哚和鸟氨酸脱羧酶试验对流感嗜血杆菌进行传统的生物学分型,分为Ⅰ～Ⅷ八个生物型。回顾患者病史资料,分析生物学分型和流感嗜血杆菌所引起的疾病之间的关系。用流感嗜血杆菌荚膜编码基因（bexA）和a—f型特异性荚膜基因设计引物,采用PCR技术对流感嗜血杆菌进行荚膜基因检测。通过生物学分型和荚膜基因分型结果的比对,探讨两者之间的关联。结果分离的100株流感嗜血杆菌生物学分型结果如下：Ⅲ型6株,Ⅳ型28株,Ⅴ型1株,Ⅵ型54株,Ⅶ型11株。未分离到Ⅰ型、Ⅱ型和Ⅷ型。分析患者的临床诊断,发现主要流行株Ⅵ型流感嗜血杆菌主要引起患者肺炎（包括支气管肺炎和新生儿肺炎）和支气管炎（包括毛细支气管炎和喘息性支气管炎）。荚膜基因分型结果显示,未分离到b型和b-型流感嗜血杆菌。共分离到1株f型,其余99株均为无荚膜抗原的不可分型流感嗜血杆菌。生物学分型和荚膜分型之间无明显的相关性。结论该院分离的流感嗜血杆菌主要为生物型Ⅵ型。回顾患者病史,发现Ⅵ型主要引起肺炎和支气管炎。荚膜基因分型显示,本地区分离的流感嗜血杆菌主要为不可分型流感嗜血杆菌。生物学分型和荚膜基因分型之间无明显相关性。     

Human lactoferrin receptor activity in non-encapsulated Haemophilus influenzae

Since the ability of bacteria to compete with lactoferrin for iron contributes to the pathogenesis of mucosal infections, the presence of lactoferrin receptor activity in non-encapsulated Haemophilus influenzae was investigated. The growth of 18 H. influenzae isolates from the sputum samples of chronic bronchitis patients and of six of seven H. influenzae throat isolates from healthy adults was stimulated by iron saturated human lactoferrin. Apo-lactoferrin did not stimulate the growth of H. influenzae. Human lactoferrin binding to iron limited bacteria was detected for 16 H. influenzae strains from chronic bronchitis patients and for five of seven isolates from healthy adults. We conclude that the majority of H. influenzae isolates tested bind human lactoferrin and that the iron from lactoferrin is used for growth.     

Survival of nontypeable Haemophilus influenzae in macrophages

In this study we have investigated the ability of nonencapsulated, nontypeable Haemophilus influenzae, NT477 to survive in the J774 mouse macrophage-like cell line. Viable, intracellular nontypeable H. influenzae could still be recovered from macrophages 72 h after phagocytosis. In contrast, H. influenzae strain Rd, an avirulent, nonencapsulated variant of a serotype d strain, was killed within 24 h. These differences suggest that NT477, in comparison to Rd, possesses unique attributes that enable it to survive in macrophages for prolonged periods. To determine whether this trait is ubiquitous amongst nontypeable H. influenzae, 33 primary clinical isolates obtained from children with otitis media were screened for their ability to survive in macrophages. Of these isolates, 82% were able to persist in an intracellular environment for periods of at least 24 h. The number of viable organisms recovered at this time ranged from 2x10(4) to 50 colony-forming units per strain indicating that the extent to which nontypeable H. influenzae can resist macrophage-mediated killing varies between strains.     

Interaction of vitronectin with Haemophilus influenzae

Eight strains of Haemophilus influenzae were tested for binding to human vitronectin. All strains adhered to vitronectin-coated glass slides but no binding was detected using soluble vitronectin, suggesting that surface association of vitronectin is a prerequisite. Vitronectin binding was not likely to be mediated by fimbriae as non-fimbriated and fimbriated isogenic strains adhered equally. Adhesion could be blocked by heparin, which is also known to block vitronectin binding to Staphylococcus aureus. However, no blocking was achieved with sialic acid-rich glycoproteins such as fetuin and mucin contrasting with Helicobacter pylori for which sialic acid seems to play an important role. With Streptococcus pneumoniae binding was detected both with soluble and surface-associated vitronectin and could not be blocked by heparin. Our results suggest that H. influenzae, Streptococcus pneumoniae and Helicobacter pylori all use distinct modes to interact with vitronectin.     

A heme-binding protein produced by Haemophilus haemolyticus inhibits non-typeable Haemophilus influenzae

Commensal bacteria serve as an important line of defense against colonisation by opportunisitic pathogens, but the underlying molecular mechanisms remain poorly explored. Here, we show that strains of a commensal bacterium, Haemophilus haemolyticus, make hemophilin, a heme-binding protein that inhibits growth of the opportunistic pathogen, non-typeable Haemophilus influenzae (NTHi) in culture. We purified the NTHi-inhibitory protein from H. haemolyticus and identified the hemophilin gene using proteomics and a gene knockout. An x-ray crystal structure of recombinant hemophilin shows that the protein does not belong to any of the known heme-binding protein folds, suggesting that it evolved independently. Biochemical characterisation shows that heme can be captured in the ferrous or ferric state, and with a variety of small heme-ligands bound, suggesting that hemophilin could function under a range of physiological conditions. Hemophilin knockout bacteria show a limited capacity to utilise free heme for growth. Our data suggest that hemophilin is a hemophore and that inhibition of NTHi occurs by heme starvation, raising the possibility that competition from hemophilin-producing H. haemolyticus could antagonise NTHi colonisation in the respiratory tract.     

Virulence of non-β-lactamase-mediated ampicilin-resistant Haemophilus influenzae

We questioned whether strains of ampicillin-resistant, non-beta-lactamase-producing (AmpR NBLP) Haemophilus influenzae with lower affinity penicillin-binding proteins (PBPs) might have altered virulence. The virulence of resistant transformant strains and the susceptible recipient was compared using infant rats. Following intraperitoneal inoculation, there was a significantly lower mortality rate and incidence and magnitude of bacteremia with two of three transformants compared to the recipient strain. Reduced virulence was not associated with greater bactericidal activity of serum or human neutrophils or faster clearance of the transformant following intravenous injection. Heated rat or human plasma supported exponential growth of the recipient, but not the transformant, suggesting deficient in vivo multiplication. We conclude that H. influenzae with altered PBPs are less virulent in an infant rat model which may be related to differences in in vivo growth.     

Binding of human hemoglobin by Haemophilus influenzae

Abstract Binding of biotinylated human hemoglobin to Haemophilus influenzae was detected when organisms were grown in heme-deplete, but not heme-replete, conditions. Hemoglobin binding was completely inhibited by a 100-fold excess of unlabelled human hemoglobin or human hemoglobin complexed with human haptoglobin. Binding was only partially inhibited by rat hemoglobin, bovine hemoglobin, human globin, and bovine globin, and not at all by heme, human serum albumin, bovine serum albumin, human transferrin, or myoglobin. Hemoglobin binding was saturable at 16–20 ng of hemoglobin per 10⁹ cfu. Binding of human hemoglobin was detected in serotypes a-f and serologically non-typable strains of H. influenzae , as well as Haemophilus haemolyticus but not Haemophilus parainfluenzae, Haemophilus aphrophilus, Haemophilus parahaemolyticus , or Escherichia coli .     

In vitro cytotoxicity of Haemophilus influenzae lipopolysaccharides for bovine aortal endothelial cells

The cytotoxicity of purified lipopolysaccharide (LPS) from a prototype Haemophilus influenzae type b (Hib) strain (Eagan) and three transformants, differing in their LPS phenotype, for bovine aortal endothelial cells (BAOEC) was investigated. All LPS preparations caused cell disruption and release of lactate dehydrogenase (LDH), an indicator of cytotoxicity, from BAOEC monolayers but to differing extents. There was no correlation between the cytotoxicity of purified Hib LPS to BAOEC monolayers and potential to cause bacteraemia in experimental animals.     

Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae

Non-typable Haemophilus influenzae is a common commensal organism in the human upper respiratory tract and an important cause of localized respiratory tract disease. The pathogenesis of disease begins with bacterial colonization of the nasopharynx, a process that involves establishment on the mucosal surface and evasion of local immune mechanisms. Under the proper circumstances, the organism spreads contiguously to the middle ear, the sinuses, or the lungs, and then stimulates a brisk inflammatory response, producing symptomatic infection. In this review, we summarize our present understanding of the molecular determinants of this sequence of events. Continued investigation of the molecular mechanism of non-typable H. influenzae pathogenicity should facilitate development of novel approaches to the treatment and prevention of H. influenzae disease.     

Construction of antibiotic resistance cassettes with multiple paired restriction sites for insertional mutagenesis of Haemophilus influenzae

Insertional mutagenesis of cloned genes coupled with site specific recombination into the genome of the parent organism is an ideal method for characterizing gene function. In this paper we describe the production and utility of two antibiotic resistance cassettes for use in Haemophilus influenzae. The mutagenic elements encode resistance to chloramphenicol or spectinomycin. Multiple paired restriction enzyme sites bound both cassettes. Use of these constructs to create mutants in H. influenzae demonstrated that the cassettes are readily incorporated into the genome in single copy and allow easy detection of mutant constructs. The insertions are stable following repeated in vitro passage. In addition, the elements are compatible with each other and allow the construction of multiple mutations within a single strain.     

Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram‐negative bacteria, a major subgroup of extracellular proteins called self‐associating autotransporters (SAATs) can mediate cell–cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae Hap_S passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X‐ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell–cell interaction. The C‐terminal SAAT domain folds into a triangular‐prism‐like structure that can mediate Hap–Hap dimerization and higher degrees of multimerization through its F1–F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell–cell interaction and formation of complex microcolonies.     

Chemical synthesis of Haemophilus influenzae glycopeptide conjugates

A simple procedure for conjugating synthetic fragments of the capsular polysaccharide of Haemophilus influenzae type b, poly-3--D-ribose-(1, 1)-D-ribitol-5-phosphate (sPRP) to linear peptides is described. The procedure consists of (i) reacting the amino group of amino-heptyl sPRP with m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) in phosphate buffer, pH 7.5; (ii) selectively coupling the MBS-modified sPRP to the sulfhydryl group of the cysteine residue of peptides containing functional T-helper cell epitope(s). The glycopeptide conjugates were purified by gel filtration chromatography, biochemically characterized, and elicited protective level of anti-PRP antibody responses in rabbits. Abbreviations: PRP, poly-3--D-ribose-(1, 1)-D-ribitol-5-phosphate; sPRP, synthetic oligo-3--D-ribose-(1, 1)-D-ribitol-5-phosphate; Hib, Haemophilus influenzae type b; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide; PEG, polyethylene glycol monomethyl ether; CRM 197, a non-toxic diphtheria toxin mutant; TT, tetanus toxoid; DT, diphtheria toxoid; OMP, outer membrane protein; RP-HPLC reverse phase high pressure liquid chromatograph     

The heme-binding lipoprotein (HbpA) of Haemophilus influenzae: role in heme utilization

Haemophilus influenzae has an absolute growth requirement for heme and a heme binding lipoprotein (HbpA) has been implicated in the utilization of this essential nutrient. HbpA was identified by examining clones from an H. influenzae genomic library that caused Escherichia coli harboring the clone to bind heme. However, HbpA has not been shown to mediate heme acquisition in H. influenzae. We constructed an insertional mutation of hbpA in a nontypeable H. influenzae strain and demonstrated a role for the gene in utilization of multiple heme sources. This is the first report confirming a role for HbpA in utilization of heme.     

Catalase as a source of both X- and V-factor for Haemophilus influenzae

Haemophilus influenzae requires two growth factors, designated factor X (porphyrin) and factor V (NAD). Mammalian catalases contain both bound heme and NADPH. This study shows that catalase can supply both factors X and V to H. influenzae in vitro, thus representing a potential in vivo source of these essential growth factors.     

Differential utilization by Haemophilus influenzae of haemoglobin complexed to the three human haptoglobin phenotypes

Haemophilus influenzae has an absolute requirement for heme, which may be supplied as the haemoglobin-haptoglobin complex. Utilization of haemoglobin-haptoglobin by H. influenzae is mediated by a family of proteins termed the haemoglobin-haptoglobin binding proteins (Hgps), of which a given strain may contain up to four genes. Human haptoglobin occurs in three phenotypes (1-1, 2-1 and 2-2). Using mutant derivatives of an H. influenzae type b strain that expressed single Hgps we analysed the ability of each Hgp to utilize haemoglobin complexed to the various haptoglobin phenotypes. A strain expressing only HgpB was able to utilize haemoglobin bound to all haptoglobin phenotypes significantly better than strains expressing either HgpA or HgpC.     

儿童肺炎链球菌和流感嗜血杆菌耐药及感染特征分析

目的 了解儿童呼吸道感染肺炎链球菌(Streptococcus pneumonia,SP)和流感嗜血杆菌(Haemophilus influenzae,Hi)的分布特征、耐药情况,及耐药菌抗生素间的相互影响,以更合理地指导临床用药.方法 对2009-2010年临床呼吸道感染患儿进行痰、咽拭子或肺泡灌洗液培养分离Hi和SP.因子需求试验鉴定Hi,头孢硝噻酚法检测β-内酰胺酶;奥普托辛和胆汁溶菌试验确认SP.两菌均采用K-B法检测常用对抗生素的耐药性.结果 收集SP 495株,Hi 515株,多见于3岁以下儿童,以呼吸科分离率最高.SP对红霉素、四环素、阿奇霉素、复方新诺明的耐药率分别为98.4％、66.1％、98％和81.6％,其对青霉素敏感性仅为9.5％.Hi有42.7％产生β-内酰胺酶,Hi对氯霉素、复方新诺明、氨苄青霉素、阿奇霉素的耐药率分别为22.1％、21.6％、36.7％和62.7％.与青霉素敏感SP和β-内酰胺酶阴性Hi相比,耐药SP和阳性Hi更易对氯霉素、四环素和复方新诺明耐药.结论 SP和Hi以婴幼儿为主,多见于呼吸科.其耐药情况严峻,青霉素耐药和产β-内酰胺酶菌株会诱导其他抗生素耐药,引发多重耐药.合理使用抗生素以及对两菌的耐药监测应引起高度重视.     

Phase variation of Haemophilus influenzae lipopolysaccharide: Characterization of lipopolysaccharide from individual colonies

Abstract The lipopolysaccharide (LPS) of Haemophilus influenzae expresses a number of core oligosaccharide epitopes on its outer surface. The expression of individual epitopes is subject to frequent (approximately 1% bacteria/generation) reversible phase variation, as determined by colony immunoblots. We have used a microtechnique for the extraction of LPS from individual colonies, whose LPS antigenic phenotype has been identified, so that the LPS can be studied by tricine sodium dodecylsulphate polyacrylamide gel electrophoresis (T-SDS-PAGE). This avoids the introduction of heterogenous phase-varying LPS which is inevitable if bacteria from colonies are grown in broth culture prior to LPS extraction and analysis. Using these techniques we have investigated the repertoire of LPS phase variation exhibited by H. influenzae strain RM7004 (a serotype b meningitis isolate). This technique will facilitate the study of bacteria in which there is variable LPS expression.     

ELISA方法测定血清中Hib多糖抗体含量的条件比较研究

分别以牛血清白蛋白和人血清白蛋白作为封闭液的主要成分测定7份血清中抗b型流感嗜血杆菌荚膜多糖的抗体含量,结果显示,两组数据之间没有显著性差异(P>0.05)。分别用HRP标记的羊抗人IgG和HRP标记的鼠抗人IgG测定该7份血清,结果显示两组结果无显著性差异(P>0.05)。同时,将血清反应的温度和时间由37℃1.5h改为4℃16h(过夜),结果显示两者无显著性差异(P>0.05)。从而优化了该方法。