A Mutation in the sap Operon Attenuates Survival of Nontypeable Haemophilus influenzae in a Chinchilla Model of Otitis Media

Abstract

Bacteria have evolved strategies to resist killing by antimicrobial peptides (APs), important effectors of innate immunity. The sap (sensitivity to antimicrobial peptides) operon confers resistance to AP-mediated killing of Salmonella. We have recently shown that sapA gene expression is upregulated in the middle ear in a chinchilla model of nontypeable Haemophilus influenzae (NTHI)-induced otitis media. Based on these findings, we constructed an NTHI strain containing a Lux reporter plasmid driven by the sapA promoter and demonstrated early yet transient expression of the sap operon within sites of the chinchilla upper airway upon infection. We hypothesized that the sap operon products mediate NTHI resistance to APs. In order to test this hypothesis, we constructed a nonpolar mutation in the sapA gene of NTHI strain 86-028NP, a low-passagenumber clinical isolate. The sapA mutant was approximately eightfold more sensitive than the parent strain to killing by recombinant chinchilla ␤-defensin 1. We then assessed the ability of this mutant to both colonize and cause otitis media in chinchillas. The sapA mutant was significantly attenuated compared to the parent strain in its ability to survive in both the nasopharynx and the middle ear of the chinchilla. In addition, the mutant was impaired in its ability to compete with the parent strain in a dual-strain challenge model of infection. Our results indicate that the products of the sap operon are important for resisting the activity of APs and may regulate, in part, the balance between normal carriage and disease caused by NTHI.

Phosphorylcholine Decreases Early Inflammation and Promotes the Establishment of Stable Biofilm Communities of Nontypeable Haemophilus influenzae Strain 86-028NP in a Chinchilla Model of Otitis Media

“…One possible explanation of these results that we considered was a greater early killing of the mutant bacteria immediately postchallenge compared to that of the parental strain, thus resulting in increased amounts of free LOS and other inflammatory agonists. Similar increased early inflammation has been noted for defensin-sensitive mutants of NTHi (25). Thus, we compared the two strains for their relative ability to resist complement-mediated killing and the chinchilla beta-defensin cBD-1 (15).…”

“…All NTHi strains were cultivated on supplemented brain heart infusion media, which consists of brain heart infusion medium (Difco) with added NAD (Sigma) and hemin (ICN). NTHi 86-028NP is a pediatric OM isolate (2) for which virulence in the chinchilla has been extensively documented (1,19,25,26) and for which a complete genomic sequence is available (16,28). NTHi 86-028NP licD is a PCho Ϫ mutant derived from NTHi 86-028NP that has been described previously (55).…”

A Novel Putrescine Exporter SapBCDF of Escherichia coli

“…typhimurium (23) and Haemophilus influenzae (24) contribute to resistance FIGURE 1. Putrescine concentrations of culture supernatant of screened strains and depiction of putative sapABCDF operon.…”

“…This hypothesis was experimentally confirmed in H. influenzae using LL-37 and ␤-defensin (30). Furthermore, H. influenzae sapA mutant exhibited attenuated survival in a chinchilla model of otitis media (24). The amino acid identity of SapABCDF in E. coli and S. enterica sv.…”

Antimicrobial Peptide Resistance Mechanisms of Human Bacterial Pathogens

“…Genetic and biochemical evidence has demonstrated that gonococcal resistance to protegrin-1 and human cathelicidin LL-37 is modulated by overexpression of the mtr genes (Shafer et al, 1998). S. enterica mutants with a defect in the sap locus sharing homology with potassiumlinked efflux systems in E. coli are more susceptible to the cationic AMP protamine (Parra-Lopez et al, 1994), while a homologous locus in H. influenzae promotes resistance to β-defensin (Mason et al, 2005). Certain S. aureus strains harbor a multiresistance plasmid pSK1 that encodes the QacA efflux pump.…”