Characterization of lactate utilization and its implication on the physiology of Haemophilus influenzae

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 20.1 μM 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.

Outer membrane protein P1 is the CEACAM‐binding adhesin of Haemophilus influenzae

“…Chromosomal restoration of ompP1 was performed according to Lichtenegger et al . (). The ompP1 gene was linked to a kanamycin resistance gene cassette via SOEing (Splicing by Overlap Extension) PCRs.…”

A novel mechanism for the biogenesis of outer membrane vesicles in Gram-negative bacteria

“…For this purpose, respective strains were grown to an OD 490 of 0.8-1 in BHI–NAD–hemin medium supplemented with or without 100 μM 2,2′-dipyridyl in case of H. influenzae or to an OD 600 of 0.7–0.9 in LB medium in case of V. cholerae and E. coli . Bacterial RNA extraction, DNase digestion, cDNA synthesis and qRT-PCR were performed as described previously 52 . To determine the gene expression of vacJ and yrbE in H. influenzae under in vivo conditions, CD-1 mice were intranasally inoculated with Rd KW20 essentially as described previously 8 with an inoculum of approximately 5 × 10 8 c.f.u.…”

The Pseudomonas aeruginosa Complement of Lactate Dehydrogenases Enables Use of d - and l -Lactate and Metabolic Cross-Feeding

“…Previous work has indicated that phenazine-supported ATP generation and survival in anaerobic cell suspensions depend on reactions associated with pyruvate fermentation and oxidation ( 7 , 9 ). Furthermore, lactate, a product of pyruvate fermentation, is a major component of CF sputum ( 11 ) and a significant carbon and energy source for pathogens and commensals of mammalian hosts ( 12 – 15 ). These observations motivated us to investigate the roles of enzymes that interconvert pyruvate and lactate in P. aeruginosa growth and biofilm development, including a previously uncharacterized l -lactate dehydrogenase.…”