In this study, we have described the construction of a Himar1
-based transposon system for use in L. monocytogenes
. This construction decreases many of the issues observed using previous transposon systems in L. monocytogenes
, specifically, in instances of multiple transposon hops within a single bacterium, clonal populations within a given library (6
), and insertion “hot spots” (4
). Furthermore, the inclusion of T7 promoters makes negative-selection screens possible. This transposon delivery system is also effective in Bacillus anthracis
(J. Beaber, J. Zemansky, D. A. Portnoy, R. Calendar, unpublished results).
In order to identify extragenic factors involved in LLO production, activity, or secretion, 50,000 transposon insertions were screened on sheep's blood agar plates for hypohemolytic phenotypes. Critical for virulence, LLO is subject to multiple levels of regulation (68
). Given the increased complexity of Himar1 mariner
transposon mutant libraries, the likelihood of identifying extragenic regulators of LLO, and therefore likely virulence determinants, was increased. Additionally, to our knowledge, this study represents the first published screen for mutants with a hypohemolytic phenotype rather than for those with an ahemolytic phenotype. As a result of the screen, 193 mutants were initially identified as having a hypohemolytic phenotype by visual inspection (Table ). Plaque assays of murine cells were performed on the 51 mutants with the most discernible visible defect (Table ).
Given the importance of hly
in virulence, we expected that a greater fraction of our mutants would exhibit defects escaping from the primary and secondary vacuoles in the ex vivo plaque assay (68
). Curiously, of the mutants analyzed, only eight had repeatable, substantive plaque defects. One possibility for the discrepancy between blood plate phenotype and the lack of an in vivo defect may be that the phenotypes revealed on blood agar might be too subtle to be physiologically relevant. The blood plate assay is very sensitive, as zones of hemolysis are the result of discrete foci of toxin activity occurring over the course of 48 h.
The screen successfully identified both potential novel virulence factors and additional roles for previously described factors; in the eight mutants with both a blood plate phenotype and a plaque defect, six genes—yjbH
, and cggR
—have not previously been characterized for L. monocytogenes
. Although the other two genes, mprF
, are known to contribute to virulence, a potential relationship between each of these factors and the secretion of LLO has not previously been established (9
The six characterized mutants displayed a range of defects in the ex vivo plaque assay, the hemolytic-activity assay, and in vivo (Tables and ; Fig. and ). The genes ymdB (the gene containing the putative metallo-phosphoesterase domain) and ytqI (encoding the putative oligoribonuclease) have not been well characterized, and speculating about a potential role in LLO regulation is difficult. Additional work is necessary in order to determine whether the phenotypes of these mutants are the result of a direct relationship with LLO or the PrfA regulon or a more general pleiotropic effect. However, the identification of these genes does suggest that there are as-yet-unidentified factors or pathways that regulate LLO production, secretion, or activity.
More intriguing are the transposon insertions in yjbH
, and prsA2
. The association of a strain with a mutation in prpC
, the phosphatase directly upstream of the eukaryotic-kinase-type serine/threonine kinase, with a virulence defect parallels a similar defect in prpC prkC
mutants of other gram-positive pathogens (15
). Based on BLAST alignments (http://blast.ncbi.nlm.nih.gov/
), PrpC is approximately 40% identical and 60% similar to its likely homolog in each of S. pyogenes
M1 group A streptococci, S. agalactiae
group B streptococci, E. faecalis
, and S. pneumoniae
. Equally intriguing is a recent study that has suggested a role for PrkC in B. subtilis
in sensing the extracellular environment by binding fragments of peptidoglycan (72
). It is therefore tempting to ascribe a direct role for prpC
in L. monocytogenes
virulence, possibly by allowing the bacteria to “sense” its environment. However, while mutations in these genes are known to cause changes in the expression of certain virulence factors, they have also been linked to effects on growth, morphology, and cell division and other pleiotropic effects (32
). Additional work is required to discern the precise relationship between this kinase/phosphatase pair and members of the PrfA regulon. The reason a difference exists between the phenotype on the blood agar plate (Fig. ) and the results of the hemolytic-activity assay (Table ) remains unclear.
mutants displayed similar blood plate phenotypes, similar hemolytic activities, and similar virulence defects (Fig. and ; Tables and ). In B. subtilis
, their predicted homologs have recently been characterized as regulating the activity of the disulfide stress regulator Spx (41
). This transcription factor is responsible for maintaining thiol-redox homeostasis (41
), and it is tempting to speculate that the L. monocytogenes spx
(lmo2191) gene product may answer one of the long-standing questions regarding the toxin. The cholesterol-dependent cytolysins, including LLO, are also known as the “thiol-activated” cytolysins due to the presence of a conserved cysteine residue (76
). Crude preparations of these toxins are readily oxidized and require the addition of a reducing agent to reverse the effects. However, this effect diminishes as the purity of the preparation increases, and mutating the cysteine to an alanine does not eliminate LLO hemolytic activity (58
). One possible explanation for the conservation of this cysteine residue is that it serves a regulatory role and may be the target site of some external molecule. Perhaps L. monocytogenes
Spx controls a factor that binds to this residue, sequestering activity. YjbH and/or ClpX would then work to limit the repression by Spx. Consistent with this hypothesis, a transposon insertion into a gene encoding thioredoxin-like protein (lmo1609) was identified (Table ), although this mutation did not lead to a significant plaque defect.
Most interesting was our finding that a transposon insertion into prsA2 affected LLO secretion and activity. There was a distinct blood plate phenotype and plaque defect for this mutant (Fig. ; Table ). Furthermore, ΔprsA2 culture supernatant and purified, full-length LLO from a strain containing a transposon in prsA2 had similar decreased levels of hemolytic activity relative to WT levels (Table ). Finally, Western blot analysis revealed that while the total levels of LLO exported into the supernatant were similar to WT levels, there were additional lower-molecular-weight species of the toxin (Fig. ). The amounts of these species decreased in the complemented strains and drastically increased in a strain of L. monocytogenes that overproduced LLO (Fig. ). These additional bands did not stain with an anti-FLAG antibody when they were purified from a prsA2 mutant expressing LLOFLAG, indicating C-terminal cleavage (Fig. ). These results strongly suggested that PrsA2 contributes to the proper folding of extracytoplasmic LLO.
Given the additional results that strains lacking PrsA2 have diminished lecithinase activity and a diminished ability to swarm, it is likely that PrsA2 contributes to the folding of several other exported proteins. We therefore hypothesize that the decrease in LLO activity, as well as both the plaque and in vivo defects in prsA2 mutants, arises as a result of multiple misfolded virulence factors. Our lab is currently investigating this possibility.
Previous studies had identified a link between the expression of prsA2
and the master virulence transcription factor prfA
transcript levels were upregulated during intracellular growth (9
) and upregulated in bacteria grown in cytosol-mimicking medium (52
), and PrsA2 protein was upregulated in a prfA
* background (56
). Given the computationally identified PrfA box upstream of prsA2
, it was hypothesized that this gene was directly regulated by PrfA (52
). However, our results suggest that PrfA does not directly regulate prsA2
expression: a 116-bp region upstream of prsA2
lacking the identified PrfA box (Fig. ) was sufficient to complement the transposon insertion ex vivo (Table ) and the in-frame deletion both in the hemolytic-activity assay and in vivo (Fig. ). While PrfA may indirectly control prsA2
expression, there appear to be environmental conditions unassociated with virulence, such as swarming, under which prsA2
expression is independent of PrfA (Fig. ).
During infection, L. monocytogenes
undergoes dynamic changes in gene and protein expression (9
). This includes the translation of a variety of different PrfA-regulated genes within a short period of time: the genes for internalins (InlA and InlB) to promote uptake into a cell; LLO, the phospholipases C (PI-PLC and PC-PLC), and Mpl to promote phagosome escape; and then ActA to hijack host cell actin (84
). The PrfA-regulated genes comprise the most abundant secreted proteins during this transition (71
; data not shown).
Interestingly, the effect of the prsA2
mutants on secreted proteins became more pronounced the more LLO was secreted (Fig. , lanes 2, 4, and 8); the presence of the additional bands noted in the legend of Fig. is most pronounced in the LLOS44A
background, followed by the WT background and then the Δhly
background. We therefore hypothesize that L. monocytogenes
PrsA2 is necessary for the bacterium during times of increased export stress. This includes intracellular growth when multiple virulence factors—especially LLO—are produced and secreted in large quantities. Export of these factors occurs through the Sec machinery. Entry and passage through the Sec translocation channel requires substrate proteins to be in a primarily unfolded state (82
). Folding at the trans
side of the membrane is then facilitated by a number of chaperones known as the foldases and include peptidyl-prolyl cis/trans
isomerases (of which PrsA2 is a member) (82
). Accumulation of unfolded LLO in the space between the extracytoplasmic side of the membrane and the cell wall in the mutants lacking prsA2
may lead to either protein degradation or the release of misfolded toxin, resulting in the decrease in hemolysis in vitro (Fig. ; Table ) and the virulence defect in vivo (Fig. ). It is likely that other virulence factors, such as PC-PLC (Fig. ), are similarly affected, although whether these factors interact with PrsA2 directly remains unclear. Precedence for this model exists in B. subtilis
, where the expression of chaperones has been shown to increase in response to extracytoplasmic secretion stress (although those studies were performed with a mutant prsA
Our hypothesis may explain results from other studies. Gram-positive bacteria have a quality control system for exported factors within the extracytoplasmic space between the cell membrane and peptidoglycan layer (82
). One system involves the proteolytic digestion of misfolded proteins by extracytoplasmic proteases, such as the HtrA family of proteases (11
). A recent study found that L. monocytogenes
HtrA levels increased in prfA
* strains (56
), and HtrA has been shown to contribute to virulence (77
). Our hypothesis is consistent with a model that, under increased export stress, including that experienced upon infection, L. monocytogenes
upregulates the expression of several factors, including multiple external chaperones such as PrsA2 to manage this stress. These chaperones may prove to be potential targets for therapeutic interventions.