Unexpected Link between Lipooligosaccharide Biosynthesis and Surface Protein Release in Mycobacterium marinum

Aniek D Van Der Woude,Debasmita Sarkar,Apoorva Bhatt,Marion Sparrius,Susanne A Raadsen,Louis Boon,Jeroen Geurtsen,Astrid M Van Der Sar,Joen Luirink,Edith N.G Houben,Gurdyal S Besra,Wilbert Bitter

doi:10.1074/jbc.m111.336461

Abstract

The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence.

Highlights

Various cell surface proteins of pathogenic mycobacteria have been implicated in virulence
Identification of PE_PGRS Secretion Mutants Indicate Link to LOS Biosynthesis—In a previous screen, three mutants deficient for PPE41 secretion were identified in M. marinum, all with a transposon insertion in the ESX-5 cluster, two in gene eccA5, and one in mmar_2676 [9, 10]
A possible explanation for this unanticipated result is that different transposon mutagenesis studies in both M. marinum and M. tuberculosis have shown that mutants in most ESX-5 genes cannot be isolated [10, 24],4 indicating that these mutations might be lethal

Summary

Background

Various cell surface proteins of pathogenic mycobacteria have been implicated in virulence. Results: A screen for secretion defects of specific cell surface proteins in Mycobacterium marinum identified predominantly lipooligosaccharide (LOS) biosynthesis mutants. The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface These mutants had insertions in a gene cluster associated with LOS biosynthesis. ESX-5 has been shown to be responsible for the secretion of different members of the PE and PPE protein families [9, 10] These enigmatic protein families are unique to mycobacteria and highly expanded among slow growing mycobacteria, especially Mycobacterium kansasii, Mycobacterium marinum, and species of the M. tuberculosis complex [11]. This, together with the diversity and high number of the LOS biosynthesis mutants found, indicates an intriguing relation between PE_PGRS release and cell wall composition

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION