Experiments described in this study indicate that the P. aeruginosa gene PA2783 encodes a secreted endopeptidase, which we have named Mep72. The predicted protein, which has a typical leader peptide at its amino terminus, belongs to the M72 family of metallopeptidases . According to the MEROPS Peptidase Database, the P. aeruginosa Mep72 is a member of the peptidyl-Asp metallopeptidases (M72.001), proteins that degrade aspartate containing substrates by cleaving peptide bonds at the amino side of aspartate or cysteic acid . Additional experiments would be needed to confirm such an activity.
P. aeruginosa produces at least three well characterized extracellular proteases/peptidases, LasB, LasA, and PrpL. LasB is a metalloendopeptidase that belongs to the thermolysin (M4) family , LasA is a 20-kDa zinc metalloendopeptidase that belongs to the β-lytic endopeptidase family (M23) [39, 46], and PrpL is a 27-kDa endopeptidase belonging to the serine endopeptidase family [39, 47, 48]. Compared with these extracellular proteases, Mep72 has several notable characteristics. First, it is less efficient in proteolytic activity. Neither the loss of the functional gene in P. aeruginosa nor the presence of multiple copies of mep72 (pAB2) in PAO1 or PAO-R1 enhanced the proteolytic activity (data not shown). Second, similar to LasB, LasA, PrpL, and other P. aeruginosa proteases, Mep72 is likely to be secreted to the extracellular environment. The lack of transmembrane regions within the predicted protein further supports this suggestion (data not shown). The presence of LasB and other proteases within the PAO1 supernatant prevented us from detecting Mep72 proteolytic activity (data not shown). We were fortunate to detect strong extracellular proteolytic activity in E. coli DH5α carrying a mep72 plasmid (Figure 6A). However, similar to other P. aeruginosa proteins, when we overexpressed mep72 from the pBAD inducible promoter, Mep72 was trapped within the E. coli membranes (probably in inclusion bodies) (Figure 6C, D). We plan to produce polyclonal antibodies to the recombinant Mep72 encoded by pAB4 and utilize the antibodies to detect Mep72 within the supernatant of PAO1. Third, unlike LasB, LasA, and PrpL, Mep72 contains additional domains, two CHO-binding modules at the carboxy terminus region (Figure 5A). Whether the CHO-binding and the endopeptidase domains represent two separate functions of Mep72 or are required for a single target is yet to be determined. Fourth, LasB, LasA, and PrpL are among the virulence factors whose production is stringently controlled by the QS system . Since the P. aeruginosa las and rhl QS systems are controlled by Vfr, the three extracellular proteases are indirectly regulated by Vfr . In contrast, Mep72, which is directly controlled by Vfr, may not be influenced by QS systems. Through several preliminary experiments, we ruled out the possibility that mep72 expression is regulated by either the las or the rhl system (data not shown). Fifth, unlike other proteases, the impact of Mep72 on P. aeruginosa virulence is not defined yet. The loss of functional Mep72 in PAO1 did not impact the production of several virulence factors including LasB, LasA, pyocyanin, or pyoverdine (data not shown). Additionally, preliminary analysis using the murine model of thermal injury showed that the in vivo virulence of PW5661 is comparable to that of its parent strain (data not shown).
The first such endopeptidase enzyme described was isolated from Pseudomonas fragi, a pyschrotrophic, proteolytic organism that causes meat spoilage by producing a single extracellular neutral protease, endoproteinase Asp-N, at lower temperatures [50, 51]. As Mep72 has amino acid identity with the P. fragi protein in the endopeptidase region (data not shown), and since P. aeruginosa grows at 10°C, we examined the proteolytic activity of Mep72 at this temperature. At this temperature, Mep72 activity would not be masked by other P. aeruginosa extracellular proteases, which are activated at 37°C. However, we did not detect any difference in their proteolytic zones. The two CHO-binding domains carried by Mep72 belong to the CBM_4_9 family. Proteins in this family are important for very diverse CHO metabolic processes including enzymatic degradation of oligosaccharides, cellulase activity and hydrolase activity by acting on glycosyl bonds [40, 52, 53]. Whether the CBM_4_9 domain in Mep72 plays a role in P. aeruginosa binding to the alveolar mucus during lung infections is not known.
All available evidence, including data provided in this study, suggests that Vfr is a DNA-binding transcriptional regulator [13, 14, 18, 19] (Figures 2 and 7). Using qRT-PCR, we also detected transcriptional regulation of mep72 expression by Vfr (Figure 2). Additionally, one of the unique features of mep72 is its pattern of expression throughout the growth cycle of PAO1, which we detected with both lacZ and phoA translational fusions (Figures 3 and 4). In these experiments, mep72 expression was enhanced by the presence of multiple copies of vfr (lacZ) or expression the lac promoter, which is constitutively expressed in P. aeruginosa (phoA). The same pattern likely exists in PAO1 and PW5661 carrying pUCP19 (vector control); however, due to the low level of mep72 expression, we did not detect it. These results strongly suggest that the unique pattern of mep72 expression is due to the effect of Vfr-independent translational/post-translational regulation.
This pattern of expression is not a feature of the Vfr regulon. Many genes of the Vfr regulon including lasB, lasA, lasR are part of the quorum sensing system and as such, expression is induced at later rather than earlier stages of growth [16, 54]. The significance of this pattern of expression is not known at this time. However, during our analysis of the P. aeruginosa global regulator PtxR (using ptxR-lacZ transcriptional fusions), we previously reported a pattern of expression that mimics that of PA2782-mep72. The expression of one of the ptxR-promoter nested deletions reached a peak at early stage of growth, sharply declined after that, and continued a low level of expression toward the end of growth cycle . Similar to mep72, Vfr binds to the ptxR upstream and directly regulates ptxR expression .
Through the examination of the promoter regions of genes regulated by Vfr including lasR, toxA, pvdS, prpL, and algD, Kanack et al. developed a 21-bp Vfr binding consensus sequence that consist of two halves and contain several conserved nucleotides within each half . Experimental evidence revealed that changing one or more of these conserved nucleotides within the lasR or fleQ promoters affected the expression of these genes and their regulation by Vfr [16, 18, 44]. Our current analysis confirmed that Vfr specifically binds to the PA2782-mep72 promoter (Figure 7C). As with other Vfr-regulated genes, Vfr binding to the PA2782-mep72 promoter is cAMP dependent (Figure 7C). However, in contrast to all previously identified Vfr binding sites, the potential Vfr binding region within PA2782-mep72 does not contain the intact Vfr consensus sequence (Figure 7D and E). Rather, we localized Vfr binding within the PA2782-mep72 promoter to a 33-bp sequence (probe VI), which contains only 6 bp from the left half of the Vfr consensus sequence (Figure 7E). Careful examination of the sequence revealed the presence of a 5-bp imperfect inverted repeat, with two bp mismatch (underscored), at either end of the 33-bp sequence: TGGCG-N22-CGCTG (Figure 7E). Compromising either of the repeats eliminated Vfr binding (Figure 7D and E). Thus, this sequence may constitute an alternative Vfr binding site. The TGGCG-N22-CGCTG sequence overlaps the −35 region (Figure 7E). Additionally, the 33-bp sequence contains two direct repeats (TG/TG and CA/CA) (Figure 7E). Furthermore, the 33-bp sequence contains another imperfect (7/9) inverted repeat consisting of 9 bp, TGGCGCAAA-N9-TTGCCGCCA. Probe VII, which lost the ability to bind Vfr, lacks only one bp (A) from the right side of this repeat (Figure 7E). Further analysis including DNA foot printing experiments will be done to determine the exact sequence to which Vfr binds.