The M. tuberculosis gene Rv2190c is described as encoding a hypothetical protein containing an NlpC/P60 domain. Here, we have characterized the role of this gene and its protein product, showing that Rv2190c is involved in cell wall maintenance and composition, is necessary for standard growth and virulence in vivo, and is expressed, possibly surface-exposed and immunogenic in the mammalian host. These findings provide significant groundwork towards understanding the role of this “hypothetical” protein, and, importantly, suggest that Rv2190c has potential translational utility that should be further investigated.
While our data demonstrate that Rv2190c
is not essential, lack of this gene was associated with a growth defect in vitro
(), in agreement with Sassetti's findings 
. These findings are also consistent with a role for normal growth and development documented for NlpC/P60 domain-containing proteins in other bacteria, such as CwlF and LytF from Bacillus subtilis
, p75 from Lactobacillus casei
, p60 of L. monocytogenes
, and cgR_1596 and cgR_2070 of Corynebacterium glutamicum
. However, in these bacteria the effect on growth was largely due to septational changes manifested by a lack of a functional hydrolase domain, and this was not the case with the Rv2190c
mutant, as it did not show any septation defects (). This could be due to the fact that the transposon insertion in the mutant is C-terminal to the hydrolase domain containing the DCSG and GD motifs and the histidine residue. However, the growth defect implicated that hydrolysis to generate daughter cells may not be the sole function for this gene product. We have further demonstrated that this attenuated growth may be associated with the role of Rv2190c in cell wall integrity, as the M. tuberculosis Rv2190c
mutant exhibited altered PDIMs (), increased susceptibility to lysozyme (), and altered gross morphology () similar to the NlpC/P60-domain-containing proteins from M. marinum
In addition to in vitro
growth defects, the Rv2190c
mutant also displayed growth alterations in vivo
(). The mutant showed enhanced survival in the mice in comparison to the wild-type and complemented strains and was attenuated for growth in the lungs (), also similar to the results obtained by Gao et al
. for the M. marinum
orthologues in zebrafish 
. It is possible that the decreased bacterial burden of the mutant in the mouse lungs was due to a general growth defect as was observed in vitro
(); however, the mutant was not attenuated for growth in the mouse spleen, in terms of either colony forming units () or gross morphology (data not shown). This lack of attenuation in the spleen suggests that the phenotype observed in the lungs may not be due to generally defective growth. This was consistent with the findings of Cox et al.
, who showed that PDIMS determined tissue specific replication of M. tuberculosis
in mice 
. The specific mutants tested (pps
: genes that are involved in either PDIM synthesis or export) were altered for PDIM levels, showed alterations in colony surface morphology and were attenuated for growth in mouse lungs but not spleen, similar to our data.
Although the transposon insertion in our Rv2190c
mutant was downstream of the encoded NlpC/P60 domain, the function of the Rv2190c protein was altered, resulting in altered cell morphology and PDIMs. This phenotype could be due to the protein misfolding due to the insertion sequence and/or could be due to the inability of the interrupted protein to properly interact with binding partners or substrates. The complement strain was not able to restore growth fully to the wild-type level; however, gross colony morphology, lysozyme resistance, and in vivo
phenotypes of the complemented strain suggest that this Rv2190c was functionally restored in our complemented strain. Rv2190c
is not predicted to be part of an operon 
, and the functional complementation shown in most of our assays suggest that the phenotypes associated with the Rv2190c
mutant are not due to polar effects. It is possible that the intermediate in vitro
growth of the complemented strain, as well as the intermediate total time-to-death during infection of mice with this strain, could be due the presence of the mutated/misfolded Rv2190c mildly interfering with the full-length protein.
Rv2190c has been shown to be an exported protein. It has a functional signal sequence in the N-terminal and has been shown to be secreted into the cell culture filtrate of M. tuberculosis
, another commonality with quite a few of the NlpC/P60 domain containing proteins that have been shown to be secreted and immunogenic. Taken together, the data from this study and others indicate that the N-terminus likely serves as signal sequence to export the protein to the cell surface to be either anchored and/or secreted (similar to p40 and p75 of Lactobacilli), and the hydrolase domain and C-terminus engage substrates and other proteins 
. As M. tuberculosis
has five NlpC/P60 genes and five Rpfs 
, it is tempting to speculate that specific associations exist between the two, enabling functional interactions. However, unlike the Rpfs, the genes encoding the NlpC/P60 family proteins are not redundant as both Rv1477
mutants in M. marinum
mutant in M. tuberculosis
show phenotypic differences. It is possible that each protein interacts with different molecules thereby decreasing the chances of redundancy.
We have demonstrated for the first time that the hypothetical protein Rv2190c is important for M. tuberculosis growth and virulence. While not an essential protein, lack of Rv2190c weakens the bacterial cell wall, indicating that inhibition of Rv2190c may allow for better penetration of other anti-mycobacterial compounds. Thus, our results suggest that Rv2190c should be further investigated as a possible drug target. In addition, our data showing that Rv2190c is expressed and immunogenic in the human host indicate that this protein could also be further studied for translational development.