Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis

Abstract

Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophila. L. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis, Legionella adelaidensis and Legionella londiniensis, located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM. Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis. Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase-and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity.IMPORTANCE A less virulent Legionella species, L. oakridgensis, causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis. One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis, FlgM seems to be degraded by protease activities.KEYWORDS sigma-28 factor, anti-sigma factor, FliA, FlgM, flagella, Legionella oakridgensis L egionella oakridgensis is less virulent than L. pneumophila and is pathogenic for guinea pigs (1, 2). Recently, two human cases of Legionnaires' disease caused by L. oakridgensis were reported in France (3), showing that the bacteria are able to replicate inside human cells (1,(4)(5)(6)(7). Moreover, it was demonstrated that L. oakridgensis is able to multiply inside amoebae (4) and that the type IV secretion system is a key virulence factor; new virulence factors were also described (4). In general, no additional cysteine is needed to grow L. oakridgensis on agar plates and extracel-

The Flagellar Regulon of Legionella—A Review

“…FliA is a regulator that is also involved in the expression of putative virulence genes (Bruggemann et al, 2006 ; Albert-Weissenberger et al, 2010 ; Tlapak et al, 2017 ).…”

“…Moreover, the mutant has a reduced fitness potential in amoebae (Dietrich et al, 2001 ; Hammer et al, 2002 ; Heuner et al, 2002 ; Jacobi et al, 2004 ; Molofsky et al, 2005 ; Heuner and Albert-Weissenberger, 2008 ; Schulz et al, 2012 ). Likewise, a fliA mutant strain of L. oakridgensis showed a reduced fitness in its host ( Acanthamoeba lenticulata ) (Tlapak et al, 2017 ). In addition, another L. pneumophila fliA mutant strain exhibited a reduced ability to form biofilms (Mampel et al, 2006 ).…”

“…FliA is obviously a virulence factor, and target genes of fliA were investigated to understand its implication for virulence (Bruggemann et al, 2006 ; Albert-Weissenberger et al, 2010 ; Tlapak et al, 2017 ). Target genes of fliA in L. pneumophila strains include genes of the flagellar regulon (e.g., flaA and flgM ) and others (e.g., enhA and lvrA ), illustrated in Figure 4 and listed in Table 1 (Bruggemann et al, 2006 ; Albert-Weissenberger et al, 2010 ; Schulz et al, 2012 ).…”

“… * From Table S7 (Albert-Weissenberger et al, 2010 ), modified; # Homolog gene belonging to the FliA-regulon of L. oakridgensis (data from Tlapak et al, 2017 ); § Belonging to the FliA regulon of L. pneumophila Paris replicating in A. castellanii (data from Bruggemann et al, 2006 ); FC, fold-change values . …”

“…Recently, especially L. oakridgensis simplified the identification of fliA targets potentially involved in virulence (Tlapak et al, 2017 ). L. oakridgensis strains are non-flagellated, the entire flagellar regulon is missing and only homologs of FliA and FlgM are present (Figure 5 ).…”