Abstract
Haemophilus influenzae is a Gram-negative bacillus and a frequent commensal of the human nasopharynx. Earlier work demonstrated that in H. influenzae type b, l-lactate metabolism is associated with serum resistance and in vivo survival of the organism. To further gain insight into lactate utilization of the non-typeable (NTHi) isolate 2019 and laboratory prototype strain Rd KW20, deletion mutants of the l-lactate dehydrogenase (lctD) and permease (lctP) were generated and characterized. It is shown, that the apparent KM of l-lactate uptake is 20.1μM as determined for strain Rd KW20. Comparison of the COPD isolate NTHi 2019-R with the corresponding lctP knockout strain for survival in human serum revealed no lactate dependent serum resistance. In contrast, we observed a 4-fold attenuation of the mutant strain in a murine model of nasopharyngeal colonization. Characterization of lctP transcriptional control shows that the lactate utilization system in H. influenzae is not an inductor inducible system. Rather negative feedback regulation was observed in the presence of l-lactate and this is dependent on the ArcAB regulatory system. Additionally, for 2019 it was found that lactate may have signaling function leading to increased cell growth in late log phase under conditions where no l-lactate is metabolized. This effect seems to be ArcA independent and was not observed in strain Rd KW20. We conclude that l-lactate is an important carbon-source and may act as host specific signal substrate which fine tunes the globally acting ArcAB regulon and may additionally affect a yet unknown signaling system and thus may contribute to enhanced in vivo survival.
Highlights
The only known natural habitat of Haemophilus influenzae is the human nasopharynx
The lct gene cluster of E. coli is organized in an operon consisting of lctPRD, whereby lctR encodes a transcriptional factor and lctD encodes for flavin-linked l-lactate dehydrogenase (Dong et al, 1993)
Characterization of l-lactate utilization in H. influenzae for growth in complex medium
Summary
The only known natural habitat of Haemophilus influenzae is the human nasopharynx It experiences a relatively constant and stable environment, and has retained a certain degree of metabolic flexibility in the form of a limited metabolic and substrate utilization pathway (Edwards and Palsson, 1999). Previous work (Kuratana et al, 1990; Kuratana and Anderson, 1991) reported enhanced serum resistance in response to the presence of lactate in Hib. Thereby, l-lactate may act as a host factor influencing the virulence behavior of H. influenzae (Smith, 2000). To further assess l-lactate utilization and its contribution to the development of serum resistance and colonization in the nasopharyngeal region we created defined deletion mutants in arcA and the l-lactate pathway, i.e. permease (lctP; HI1218), l-lactate dehydrogenase (lctD; HI1739.1), and characterized their influence on bacterial physiology
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