Creation of an isogenic P1-deficient mutant of Haemophilus influenzae biogroup aegyptius

Segada, Lynn M.; Lesse, Alan J.

doi:10.1016/s0378-1119(97)00545-3

Cited by 7 publications

(7 citation statements)

References 30 publications

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“…In accordance with previous observations (Li et al, 2003), we found H. influenzae biogroup aegyptius strains resistant to genetic manipulation, and our attempts to construct an isogenic BPF hadA mutant were unsuccessful, also following a previously described method (Segada and Lesse, 1997). Hence, we decided to carry out our analysis with the non-pathogenic and noninvasive E. coli strain expressing HadA, which permitted us to focus exclusively on the potential role of HadA in virulence.…”

Section: Hada Mediates Bacterial Adhesion Invasion and Microcolony Fsupporting

confidence: 72%

“…Because of the similarity of HadA to TAAs, we investigated its ability to mediate in vitro bacterial adherence and invasion of Chang epithelial cells (human conjunctival cells), which represent an in vitro model to study infection of H. influenzae biogroup aegyptius (St Geme et al, 1991;Farley et al, 1992). In accordance with previous observations (Li et al, 2003), we found H. influenzae biogroup aegyptius strains resistant to genetic manipulation, and our attempts to construct an isogenic BPF hadA mutant were unsuccessful, also following a previously described method (Segada and Lesse, 1997). Hence, we decided to carry out our analysis with the non-pathogenic and noninvasive E. coli strain expressing HadA, which permitted us to focus exclusively on the potential role of HadA in virulence.…”

Section: Hada Mediates Bacterial Adhesion Invasion and Microcolony Fmentioning

confidence: 57%

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HadA is an atypical new multifunctional trimeric coiled-coil adhesin ofHaemophilus influenzaebiogroupaegyptius, which promotes entry into host cells

Serruto

Spadafina

Scarselli

et al. 2009

Cellular Microbiology

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SummaryThe Oca (Oligomeric coiled-coil adhesin) family is a subgroup of the bacterial trimeric autotransporter adhesins, which includes structurally related proteins, such as YadA of Yersinia enterocolitica and NadA of Neisseria meningitidis. In this study, we searched in silico for novel members of this family in bacterial genomes and identified HadA (Haemophilus adhesin A), a trimeric autotransporter expressed only by Haemophilus influenzae biogroup aegyptius causing Brazilian purpuric fever (BPF), a fulminant septicemic disease of children. By comparative genomics and sequence analysis we predicted that the hadA gene is harboured on a mobile genetic element unique to BPF isolates. Biological analysis of HadA in the native background was limited because this organism is not amenable to genetic manipulation. Alternatively, we demonstrated that expression of HadA confers to a non-invasive Escherichia coli strain the ability to adhere to human cells and to extracellular matrix proteins and to induce in vitro bacterial aggregation and microcolony formation. Intriguingly, HadA is predicted to lack the typical N-terminal head domain of Oca proteins generally associated with cellular receptor binding. We propose here a structural model of the HadA coiled-coil stalk and show that the N-terminal region is still responsible of the binding activity and a KGD motif plays a role. Interestingly, HadA promotes bacterial entry into mammalian cells. Our results show a cytoskeleton re-arrangement and an involvement of clathrin in the HadA-mediated internalization. These data give new insights on the structure-function relationship of oligomeric coiled-coil adhesins and suggest a potential role of this protein in the pathogenesis of BPF.

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Section: Hada Mediates Bacterial Adhesion Invasion and Microcolony Fsupporting

confidence: 72%

Section: Hada Mediates Bacterial Adhesion Invasion and Microcolony Fmentioning

confidence: 57%

HadA is an atypical new multifunctional trimeric coiled-coil adhesin ofHaemophilus influenzaebiogroupaegyptius, which promotes entry into host cells

Serruto

Spadafina

Scarselli

et al. 2009

Cellular Microbiology

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“…Like others, we have found Hae strains stubbornly intractable to genetic manipulation, and our attempts so far with the only described successful method, of Segada and Lesse (1997), have been unsuccessful. To gain insight into the possible involvement of Bpf001 in invasion of respiratory epithelium, we decided pragmatically to investigate the function of its meningococcal homologue, NMB0419.…”

Section: A Potential Role For Bpf001 In Pathogenicity Explored Via Itmentioning

confidence: 59%

Identification and characterization of genomic loci unique to the Brazilian purpuric fever clonal group of H. influenzae biogroup aegyptius: functionality explored using meningococcal homology

Farrant

Langford

et al. 2003

Molecular Microbiology

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SummaryBrazilian purpuric fever (BPF) is a fulminant septicaemic infection of young children, caused by a clonal group of strains of Haemophilus influenzae biogroup aegyptius ( Hae ), an organism previously solely associated with conjunctivitis. Their special capacity to invade from the initial site of conjunctival infection is unexplained. A polymerase chain reaction (PCR)-amplified subtractive hybridization technique was used to identify genes specific to the BPF clonal group. A copy of bacteriophage HP1 and 46 further chromosomal loci were identified in the BPF but not in the conjunctivitis strain of Hae . Sixteen were characterized further, and one -encoding an analogue of the Legionella pneumophila epithelial cell entry-enhancing protein EnhC -was investigated in depth. Two genes, bpf001 and bpf002 , unique to the BPF clonal group were identified between homologues of HI1276 and HI1277 in a complex locus close to H. influenzae genetic island 1, recently identified in pathogenic H. influenzae type b. Bpf001 encodes a protein homologous to EnhC and to the previously uncharacterized product of the meningococcal gene NMB0419 . Functional studies of bpf001 proving intractable, NMB0419 was chosen as a surrogate for investigation and shown to modulate bacterial interaction with monolayers of human respiratory epithelial cells, promoting invasion, the first stage (for Hae ) in the pathogenesis of BPF.

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“…Owing to difficulties transforming NTHi strains by the method detailed above, several of these strains were transformed using the same P5 construct, but a method described by Segada and Lesse (1997). Subsequent analysis of mutants was as described above.…”

Section: Transformation Of Nthi Strainsmentioning

confidence: 99%

“…First, the P5 gene was inactivated using the Rd P5 Kan construct. The whole Rd P5 gene including 300 bases upstream and downstream obtained by PCR was used to replace the inactivated NTHi P5 gene by allelic replacement using the method of Segada and Lesse (1997). The NT2 recombinants expressing the Rd P5 protein were isolated by positive selection for this P5 expression by exploiting its soluble receptor-binding property.…”

Section: Introduction Of Rd P5 Into An Nthi Strainmentioning

confidence: 99%

The variable P5 proteins of typeable and non‐typeable Haemophilus influenzae target human CEACAM1

Hill

Toleman

Evans

et al. 2001

Molecular Microbiology

105

115

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SummaryHaemophilus influenzae, a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We have recently shown that numerous strains of capsulate (typeable) and acapsulate (non-typeable) H. influenzae target the carcinoembryonic antigen (CEA) family of cell adhesion molecules (CEACAMs). Moreover, the ligands appeared to be antigenically variable and, when using viable typeable bacteria, their adhesive functions were inhibited by the presence of capsule. In this report, we show that the antigenically variable outer membrane protein, P5, expressed by typeable and non-typeable H. influenzae targets human CEA-CAM1. Variants and mutants lacking the expression of P5 of all strains tested were unable to target purified soluble receptors. A non-typeable strain that did not interact with CEACAM1 was made adherent to both the soluble receptors and CEACAM1-transfected Chinese hamster ovary cells by transformation with the P5 gene derived from the adherent typeable strain Rd. However, several H. influenzae mutants lacking P5 expression continued to bind the cell-bound CEACAM1 receptors. These observations suggest that (i) CEACAM1 alone can support P5 interactions and (ii) some strains contain additional ligands with the property to target CEACAM1 but require the receptor in the cellular context. The identification of a common ligand in diverse strains of H. influenzae and the presence of multiple ligands for the same receptor suggests that targeting of members of the CEACAM family of receptors may be of primary significance in colonization and pathogenesis of H. influenzae strains.

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Creation of an isogenic P1-deficient mutant of Haemophilus influenzae biogroup aegyptius

Cited by 7 publications

References 30 publications

HadA is an atypical new multifunctional trimeric coiled-coil adhesin ofHaemophilus influenzaebiogroupaegyptius, which promotes entry into host cells

HadA is an atypical new multifunctional trimeric coiled-coil adhesin ofHaemophilus influenzaebiogroupaegyptius, which promotes entry into host cells

Identification and characterization of genomic loci unique to the Brazilian purpuric fever clonal group of H. influenzae biogroup aegyptius: functionality explored using meningococcal homology

The variable P5 proteins of typeable and non‐typeable Haemophilus influenzae target human CEACAM1

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