LytM Proteins Play a Crucial Role in Cell Separation, Outer Membrane Composition, and Pathogenesis in Nontypeable Haemophilus influenzae
Authors: Ercoli Giuseppe , Tani Chiara , Pezzicoli Alfredo , Vacca Irene , Martinelli Manuele , Pecetta Simone , Petracca Roberto , Rappuoli Rino , Pizza Mariagrazia , Norais Nathalie , Soriani Marco , Aricò Beatrice 
Published: February 2015

LytM proteins belong to a family of bacterial metalloproteases. In Gram-negative bacteria, LytM factors are mainly reported to have a direct effect on cell division by influencing cleavage and remodeling of peptidoglycan. In this study, mining nontypeable
Haemophilus influenzae
(NTHI) genomes, three highly conserved open reading frames (ORFs) containing a LytM domain were identified, and the proteins encoded by the ORFs were named YebA, EnvC, and NlpD on the basis of their homology with the
Escherichia coli
proteins. Immunoblotting and confocal analysis showed that while NTHI NlpD is exposed on the bacterial surface, YebA and EnvC reside in the periplasm. NTHI Δ
yebA
and Δ
nlpD
deletion mutants revealed an aberrant division phenotype characterized by an altered cell architecture and extensive membrane blebbing. The morphology of the Δ
envC
deletion mutant was identical to that of the wild-type strain, but it showed a drastic reduction of periplasmic proteins, including the chaperones HtrA, SurA, and Skp, and an accumulation of β-barrel-containing outer membrane proteins comprising the autotransporters Hap, IgA serine protease, and HMW2A, as observed by proteomic analysis. These data suggest that EnvC may influence the bacterial surface protein repertoire by facilitating the passage of the periplasmic chaperones through the peptidoglycan layer to the close vicinity of the inner face of the outer membrane. This hypothesis was further corroborated by the fact that an NTHI
envC
defective strain had an impaired capacity to adhere to epithelial cells and to form biofilm. Notably, this strain also showed a reduced serum resistance. These results suggest that LytM factors are not only important components of cell division but they may also influence NTHI physiology and pathogenesis by affecting membrane composition.

IMPORTANCE
Nontypeable
Haemophilus influenzae
(NTHI) is an opportunistic pathogen that colonizes the human nasopharynx and can cause serious infections in children (acute otitis media) and adults (chronic obstructive pulmonary disease). Several virulence factors are well studied, but the complete scenario of NTHI pathogenesis is still unclear. We identified and characterized three NTHI LytM factors homologous to the Escherichia coli LytM proteins. Although LytM factors are reported to play a crucial role in the cell division process, in NTHI they are also involved in other bacterial functions. In particular, YebA and NlpD are fundamental for membrane stability: indeed, their absence causes an increased release of outer membrane vesicles (OMVs). On the other hand, our data suggest that EnvC could directly or indirectly affect peptidoglycan permeability and consequently, bacterial periplasmic and outer membrane protein distribution. Interestingly, by modulating the surface composition of virulence determinants, EnvC also has an impact on NTHI pathogenesis.