BACKGROUND
the vast increase in knowledge that has accompanied the discovery of microbial pattern recognition receptors has focussed research into the microbial ligands that initiate these cellular responses  <cit>  for example it is now known that bacterial lps triggers responses via toll like receptor   <dig>  and flagellin via tlr <dig>  <cit> .
it is also increasingly appreciated that receptors may co-operate to recognise specific ligands  <cit> .
thus triacylated lipopeptide is recognised by a heterodimer of tlr <dig> and  <dig>  with diacylated lipopeptide being recognised by the tlr2/ <dig> heterodimer  <cit> .
many types of pathogens produce lipoproteins and are thus in part recognised by tlr <dig>  <cit> .
mycobacterium tuberculosis has over  <dig> probable or known lipoproteins, many of which are concentrated in the cell wall  <cit> .
whilst a role has been assigned to some of these proteins , most have not been assigned a function.
they are characterised by an acylated n-terminus, processing of which is mediated by the consecutive activity of prolipoprotein diacylglyceryl transferase  and lipoprotein signal peptidase   <cit> .
deletion of lspa reduces the virulence of m. tuberculosis.
in addition many of the lipoproteins have been found to be targets of both the innate and acquired immune response.
a prominent target of the innate response is the  <dig> kda lipoprotein encoded by rv <dig>  this molecule has been intensively researched because of its pleiotropic effects on the innate immune response that include induction of cytokine genes, bacterial killing, induction of apoptosis, and the downregulation of interferon-γ  induced mhc class ii expression  <cit> .
more recently it has also been suggested that the  <dig> kda protein acts an adhesin  <cit> .
many of the above studies of the  <dig> kda were performed with purified or recombinant protein that may not fully reflect the role of the molecule in the context of natural infection.
in particular expression in e. coli is unlikely to reproduce native patterns of post-translational modiufication.
we have previously reported the effect of deletion and overexpression of the  <dig> kda on the innate immune response  <cit> .
we found that the deletion mutant  was moderately impaired in its ability to multiply in human monocyte-derived macrophages .
surface expression of mhc class ii molecules was reduced in phagocytes infected with mtb; this effect was not seen in cells infected with Δ <dig>  Δ <dig> induced lower il-1β secretion from monocytes and mdm.
overexpression of the  <dig> kda increased il-1β, il-12p <dig> and tnf-α secretion irrespective of phagocyte maturity.
these findings confirmed the  <dig> kda protein to be an important mediator of the innate immune response in the context of the whole bacillus.
in addition to being acylated, the  <dig> kda protein is glycosylated  <cit> .
earlier work in our laboratories established that poly threonine motifs towards the n-terminal of the molecule form a major glycosylation site  <cit> .
the aim of this study was therefore to evaluate the innate immune response to Δ <dig> mutants that had been complemented with a single copy of mutagenised  <dig> kda molecules lacking the motifs for acylation and o-glycosylation respectively.
methods
generation of recombinant strains of m. tuberculosis
the  <dig> kda gene was deleted from m. tuberculosis  h37rv to produce the Δ <dig> strain as previously described  <cit> .
complementation of the Δ <dig> strain by the native and modified   <dig> kda genes led to the strains Δ19:: <dig>  Δ19::19na and Δ19::19nog.
for complementation, the native sequence  was amplified by pcr from h37rv dna.
the site-directed mutagenised genes were amplified from previous episomal constructs  <cit>  engineered to come under the control of the endogenous  <dig> kda promoter.
complementation was performed using the integrating vector pkinta, based on the l <dig> phage integration system  <cit> , which reintroduces a single copy of the  <dig> kda gene into the chromsome under the control of its own promoter at the attb site  <cit> .
pcr was used to confirm deletion and insertion as previously described  <cit>  and sequencing of the pkinta insert confirmed nucleotide differences that would result in substitution of the n-terminal cysteine residue of the mature wild-type protein with alanine in Δ19::19na; and substitution of two threonine clusters  by valine residues in Δ19::19nog.
for western blotting supernatants and sonicated preparations of wild-type m. tuberculosis h37rv and the deleted and complemented strains were fractionated by sds-page and expression of the  <dig> kda antigen compared by western blot analysis using a polyclonal anti- <dig> kda serum.
isolation and culture of monocytes
buffy coats from healthy donors were obtained from the national blood transfusion service .
following dilution in rpmi , peripheral blood mononuclear cells  were separated by centrifugation over ficoll-paque plus .
cells were washed in rpmi and counted.
cells were suspended at  <dig>  × 107/ml in rpmi/10% fcs medium and aliquots of  <dig> mls were added to  <dig> cm <dig> tissue culture flasks.
flasks were placed flat in a 5% co <dig> incubator and monocytes allowed to adhere for  <dig> h at 37°c.
non-adherent cells were removed by washing  <dig> times with  <dig> mls of pre-warmed rpmi.
finally,  <dig> mls of ice-cold pbs was added and the flasks were incubated at 4°c for  <dig> mins.
using a scraper, monocytes were gently dislodged from the bottom of the flasks and pooled in a  <dig> ml falcon tube to count.
cells were plated in rpmi containing 10% serum at 106/well in a 24-well tissue culture plate, and cultured overnight before infection.
infection of cells
bacilli used to infect cells were grown in middlebrook 7h <dig> broth supplemented with adc to mid-log phase  then frozen in aliquots in 15% glycerol.
the cfu content of aliquots was determined by serial dilution and plating on middlebrook 7h <dig> agar supplemented with oadc.
monocytes were infected at a multiplicity of infection of 1: <dig> without removing non-phagocytosed bacteria.
culture duration was  <dig> hrs., at which time supernatants were aspirated,  <dig>  μm filtered, and stored at -80°c pending analysis by elisa.
elisa
cytokine elisa was performed using the duoset elisa development systems  following the manufacturer's recommendations.
the sensitivity of the assays was  <dig> pg/ml for il-12p <dig>   <dig> pg/ml for il-1β and  <dig> pg/ml for tnf-α.
histone associated dna fragments, released into tissue culture supernatant and interpreted as evidence of apoptotic cell death, were assayed by the cell death detection elisa  according to the manufacturer's instructions.
sequence analysis
homologues of the m. tuberculosis  <dig> kda gene lpqh were identified by blast searches of sequenced genomes  <cit> .
alignment of protein sequences was performed using clustal w and results are displayed as a sequence pile-up and as a neighbour-joining tree.
strains and genome accession numbers: m. tuberculosis h37rv, al <dig> ; m. smegmatis mc <dig>  cp <dig> ; m. ulcerans agy <dig>  cp <dig> ; m. marinum m, cp <dig> ; m. leprae tn, al <dig> ; m. avium subsp.
paratuberculosis k- <dig>  ae <dig> ; m. abscessus, cu <dig> ; nocardia farcinica ifm  <dig>  ap <dig> ; rhodococcus sp.
rha <dig>  cp <dig> .
statistical methods
paired and unpaired parametric variables were compared by student's t-test.
paired and unpaired non-parametric variables were compared by wilcoxon signed rank or mann whitney u test respectively.
significance was inferred for p values ≤  <dig> .
RESULTS
bioinformatic analysis of  <dig> kda genes in various mycobacteria
the  <dig> kda or lpqh lipoprotein of m. tuberculosis belongs to a family of conserved proteins that is ubiquitous through the mycobacteria and is also found in the closely related nocardia farcinica and rhodococcus but not in other high gc gram positive bacteria such as streptomyces and corynebacteria.
in addition to the lpqh gene, m. tuberculosis possesses a paralogous gene encoding the lipoprotein lppe.
other mycobacteria have varying numbers of  <dig> kda gene homologs with the fast-growing m. abscessus possessing  <dig> paralogous genes.
figure  <dig> shows an alignment of twenty seven  <dig> kda family proteins identified from genome sequencing projects.
displayed as a neighbour-joining tree, it is apparent that the  <dig> kda proteins fall into three general sub-families: lpqh-like proteins, lppe-like proteins and a third subfamily that we term lp <dig> .
all except one protein  contain a predicted secretion signal sequence with the n-terminus of mature proteins containing a cysteine residue.
twenty-one out of twenty-six predicted full-length  <dig> kda proteins including the m. tuberculosis lpqh and lppe proteins, comply with the lipobox consensus acylation motif  <cit> .
this is consistent with the approximately 75% predictive value of the lipobox based on experimental evidence of known prokaryote lipoproteins.
cysteine residues at positions  <dig> and  <dig>  and phenylalanine at position  <dig> are conserved throughout the family.
strongly and weakly conserved groups of amino acids are also highlighted in figure 2b.
o-glycosylation does not occur at a particular motif of amino acids but occurs at specific residues, generally threonine and serine.
the m. tuberculosis lpqh  <dig> kda protein is glycosylated at a triplet and a pair of threonines at positions 14– <dig>  and 19– <dig>  <cit> .
threonine pairs are also found in several other  <dig> kda family proteins including, for example, the predicted protein from n. farcinica which has two pairs of threonine residues at positions 11– <dig> and 15– <dig>  in addition, many of the  <dig> kda homologs have n-terminal regions of the mature protein that are rich in serine residues which may be indicative of glycosylation.
taken together, it seems likely that n-terminal glycosylation and acylation are general features of the  <dig> kda protein family.
the broad distribution of this family across mycobacteria and closely related genera suggests that these lipoproteins fulfil some conserved physiological function which at present remains largely unknown.
to screen for a possible role for the  <dig> kda lipoprotein in mycobacterial physiology, we therefore generated a deletion mutant lacking the  <dig> kda molecule and complemented this mutant with the wild type and site-mutagenised copies of the  <dig> kda molecule.
generation and characterization of recombinant m. tuberculosis strains
pcr analysis showed rv <dig> to be absent from Δ <dig> and that this sequence had been successfully reintroduced into strains Δ19:: <dig> , Δ19::19na, and Δ19::19nog .
western blotting of cellular pellet indicated that the  <dig> kda was not produced in Δ <dig> .
expression of native protein of the same mw was restored close to normal levels by reintroduction of the  <dig> kda gene in strain Δ19:: <dig> .
<dig> kda protein was only detected in the supernatant of cultures of the non-acylated  and non-o-glycosylated complemented strains and was of slightly lower mw than the native  <dig> kda.
in middlebrook 7h <dig> broth the growth rate of the Δ <dig>  Δ19:: <dig>  Δ19::19na, and Δ19::19nog strains was identical .
cytokine secretion
human monocytes were infected with equal numbers of bacilli  and co-cultured for  <dig> hours.
during this period, the median secretion of il-1β was significantly reduced by deletion of the  <dig> kda gene .
introduction of the native  <dig> kda gene as Δ19:: <dig> restored secretion to wild type levels but the response to Δ19::19na and Δ19::19nog remained significantly less when compared to Δ19:: <dig> .
there was no difference between h37rv, Δ <dig> and Δ19:: <dig> in their ability to elicit il-12p <dig> or tnf from monocytes .
although the response to both the Δ19::19na and Δ19::19nog strains tended to be lower, these differences were also not significantly different from h37rv.
induction of apoptosis
culture supernatants from  <dig> donors were also assayed for the presence of histone associated dna fragments, a marker of apoptosis.
results for each subject were normalised to unstimulated cells to generate an enrichment factor.
the Δ <dig> and Δ19::19na and Δ19::19nog were associated with lower levels than h37rv or the Δ19:: <dig> strain.
however responses varied considerably between donors and none of these trends attained statistical significance .
discussion
we have investigated deletion of the  <dig> kda lipoprotein  from m. tuberculosis and chromosomal complementation of the deletion mutant by the wild type gene and site directed mutagenised variants lacking motifs for acylation and o-glycosylation.
we have determined that both acylation and o-glycosylation are necessary for the protein to remain within the cell.
consistent with our previous findings, the  <dig> kda is an important stimulus for the production of pro-inflammatory il-1β, an effect that is dependent on acylation and o-glycosylation.
the effect of deletion and complementation on il-12p <dig> and tnf secretion was less marked with no statistically significant differences between strains.
although deletion of the  <dig> kda reduced apoptosis, an effect that could also only be reversed by complementation with the wild type gene, the results were variable between donors and did not attain statistical significance.
an interesting finding was that  <dig> kda protein was only detected in the supernatant of cultures of the non-acylated  and non-o-glycosylated complemented strains, whereas the Δ19:: <dig> strain expressed the molecule in both pellet and supernatant.
this suggests that in order to be retained within the cell wall both acylation and glycosylation are necessary for anchoring within the cell wall.
whether this relates to a specific physicochemical interaction or merely reflects the recognised hydrophobicity of the mycobacterial cell membrane remains to be determined.
sartain and belisle have recently shown that o-glycosylation affects the positioning in the cell wall but not the enzymatic activity of the superoxide dismuase sodc  <cit> .
in a previous study overexpression of the  <dig> kda in m. smegmatis reduced its capacity to induce the secretion of il-12p <dig> and tnf <cit> .
this effect was dependent on acylation and glycosylation, as tranformation of, m. smegmatis with na and nog variants of the  <dig> kda did not reduce the secretion of these cytokines.
by contrast overexpression of the native  <dig> kda molecule in Δ <dig> strain of virulent m. tuberculosis had precisely the opposite effect, with the production of il-12p <dig> and tnf increased irrespective of phagocyte maturity  <cit> .
in this study we reintroduced the  <dig> kda gene as a single copy into the chromosome of h37rv under the control of its own promoter.
we precisely reproduced our previous findings with respect to the effect of deletion of the  <dig> kda on the cytokine response of monocytes.
we have shown that the  <dig> kda mediated induction of il-1β is dependent on acylation and glycosylation.
taken together these and other studies suggest a consistent effect of acylation and o-glycosylation on the cytokine response to the  <dig> kda, but that the genetic background and level of expression are also important.
further evidence in favour of this hypothesis is our recent finding that a naturally occuring m. tuberculosis strain that lacks the  <dig> kda gene does not have the same in vitro phenotype as the engineered knock out on the rv background .
this potentially important finding requires further investigation as much of our knowledge about gene function in m. tuberculosis is inferred from studies of isogenic mutants on the h37rv background.
considerable evidence now points to the protective role of macrophage apoptosis in tuberculosis.
apoptosis may prevent the release of intracellular components and the spread of mycobacterial infection by sequestering the pathogens within apoptotic bodies  <cit> .
in addition, uptake of apoptotic debris by competent phagocytes allows efficient cross-presentation of m. tuberculosis antigens  <cit> .
thus, the avoidance of apoptosis may be considered a virulence mechanism and a recent study has in fact reported a inverse relationship between the intracellular growth rate and the ability of strains to induce apoptosis  <cit> .
two previous studies have implicated the  <dig> kda as pro-apoptotic  <cit>  and our results, although variable between donors tend to support this conclusion.
however the dependence or otherwise on post-translation modification requires additional work as the findings of lopez et al.
suggested that this effect was acylation independent, whereas the trend in our study suggest acylation is necessary .
CONCLUSIONS
in conclusion we have presented further evidence of the role of the  <dig> kda as a key modulator of the human innate immune response.
there is considerable evidence that the protein downregulates ifn-γ induced macrophage activation, an effect that will tend to favour bacillary survival during the development of an acquired immune response.
on the other hand the molecule will tend to give away the presence of bacilli to the innate system early in infection, perhaps teleologically explaining why it is not upregulated early after infection  <cit> .
in addition, this work provides further evidence of the utility of defined mutants to delineate key determinants of the innate immune response in the context of whole bacilli.
authors' contributions
kaw, rjw, grs and dby designed the research.
rjw, grs and sms derived the recombinant strains using constructs designed and prepared by on and j-lh.
kaw and smn performed and interpreted the immunological studies.
grs performed the bioinformatic analysis.
all authors contributed to analysis and to writing the manuscript.

 the vast increase in knowledge that has accompanied the discovery of microbial pattern recognition receptors has focussed research into the microbial ligands that initiate these cellular responses for example it is now known that bacterial lps triggers responses via toll like receptor and flagellin via tlr. it is also increasingly appreciated that receptors may co-operate to recognise specific ligands. thus triacylated lipopeptide is recognised by a heterodimer of tlr and with diacylated lipopeptide being recognised by the tlr2/ heterodimer. many types of pathogens produce lipoproteins and are thus in part recognised by tlr. mycobacterium tuberculosis has over probable or known lipoproteins, many of which are concentrated in the cell wall. whilst a role has been assigned to some of these proteins, most have not been assigned a function. they are characterised by an acylated n-terminus, processing of which is mediated by the consecutive activity of prolipoprotein diacylglyceryl transferase and lipoprotein signal peptidase. deletion of lspa reduces the virulence of m. tuberculosis. in addition many of the lipoproteins have been found to be targets of both the innate and acquired immune response. a prominent target of the innate response is the kda lipoprotein encoded by rv this molecule has been intensively researched because of its pleiotropic effects on the innate immune response that include induction of cytokine genes, bacterial killing, induction of apoptosis, and the downregulation of interferon-γ induced mhc class ii expression. more recently it has also been suggested that the kda protein acts an adhesin. many of the above studies of the kda were performed with purified or recombinant protein that may not fully reflect the role of the molecule in the context of natural infection. in particular expression in e. coli is unlikely to reproduce native patterns of post-translational modiufication. we have previously reported the effect of deletion and overexpression of the kda on the innate immune response. we found that the deletion mutant was moderately impaired in its ability to multiply in human monocyte-derived macrophages. surface expression of mhc class ii molecules was reduced in phagocytes infected with mtb; this effect was not seen in cells infected with Δ Δ induced lower il-1β secretion from monocytes and mdm. overexpression of the kda increased il-1β, il-12p and tnf-α secretion irrespective of phagocyte maturity. these findings confirmed the kda protein to be an important mediator of the innate immune response in the context of the whole bacillus. in addition to being acylated, the kda protein is glycosylated. earlier work in our laboratories established that poly threonine motifs towards the n-terminal of the molecule form a major glycosylation site. the aim of this study was therefore to evaluate the innate immune response to Δ mutants that had been complemented with a single copy of mutagenised kda molecules lacking the motifs for acylation and o-glycosylation respectively. methods generation of recombinant strains of m. tuberculosis the kda gene was deleted from m. tuberculosis h37rv to produce the Δ strain as previously described. complementation of the Δ strain by the native and modified kda genes led to the strains Δ19:: Δ19::19na and Δ19::19nog. for complementation, the native sequence was amplified by pcr from h37rv dna. the site-directed mutagenised genes were amplified from previous episomal constructs engineered to come under the control of the endogenous kda promoter. complementation was performed using the integrating vector pkinta, based on the l phage integration system, which reintroduces a single copy of the kda gene into the chromsome under the control of its own promoter at the attb site. pcr was used to confirm deletion and insertion as previously described and sequencing of the pkinta insert confirmed nucleotide differences that would result in substitution of the n-terminal cysteine residue of the mature wild-type protein with alanine in Δ19::19na; and substitution of two threonine clusters by valine residues in Δ19::19nog. for western blotting supernatants and sonicated preparations of wild-type m. tuberculosis h37rv and the deleted and complemented strains were fractionated by sds-page and expression of the kda antigen compared by western blot analysis using a polyclonal anti- kda serum. isolation and culture of monocytes buffy coats from healthy donors were obtained from the national blood transfusion service. following dilution in rpmi, peripheral blood mononuclear cells were separated by centrifugation over ficoll-paque plus. cells were washed in rpmi and counted. cells were suspended at × 107/ml in rpmi/10% fcs medium and aliquots of mls were added to cm tissue culture flasks. flasks were placed flat in a 5% co incubator and monocytes allowed to adhere for h at 37°c. non-adherent cells were removed by washing times with mls of pre-warmed rpmi. finally, mls of ice-cold pbs was added and the flasks were incubated at 4°c for mins. using a scraper, monocytes were gently dislodged from the bottom of the flasks and pooled in a ml falcon tube to count. cells were plated in rpmi containing 10% serum at 106/well in a 24-well tissue culture plate, and cultured overnight before infection. infection of cells bacilli used to infect cells were grown in middlebrook 7h broth supplemented with adc to mid-log phase then frozen in aliquots in 15% glycerol. the cfu content of aliquots was determined by serial dilution and plating on middlebrook 7h agar supplemented with oadc. monocytes were infected at a multiplicity of infection of 1: without removing non-phagocytosed bacteria. culture duration was hrs., at which time supernatants were aspirated, μm filtered, and stored at -80°c pending analysis by elisa. elisa cytokine elisa was performed using the duoset elisa development systems following the manufacturer's recommendations. the sensitivity of the assays was pg/ml for il-12p pg/ml for il-1β and pg/ml for tnf-α. histone associated dna fragments, released into tissue culture supernatant and interpreted as evidence of apoptotic cell death, were assayed by the cell death detection elisa according to the manufacturer's instructions. sequence analysis homologues of the m. tuberculosis kda gene lpqh were identified by blast searches of sequenced genomes. alignment of protein sequences was performed using clustal w and results are displayed as a sequence pile-up and as a neighbour-joining tree. strains and genome accession numbers: m. tuberculosis h37rv, al; m. smegmatis mc cp; m. ulcerans agy cp; m. marinum m, cp; m. leprae tn, al; m. avium subsp. paratuberculosis k- ae; m. abscessus, cu; nocardia farcinica ifm ap; rhodococcus sp. rha cp. statistical methods paired and unpaired parametric variables were compared by student's t-test. paired and unpaired non-parametric variables were compared by wilcoxon signed rank or mann whitney u test respectively. significance was inferred for p values ≤.  bioinformatic analysis of kda genes in various mycobacteria the kda or lpqh lipoprotein of m. tuberculosis belongs to a family of conserved proteins that is ubiquitous through the mycobacteria and is also found in the closely related nocardia farcinica and rhodococcus but not in other high gc gram positive bacteria such as streptomyces and corynebacteria. in addition to the lpqh gene, m. tuberculosis possesses a paralogous gene encoding the lipoprotein lppe. other mycobacteria have varying numbers of kda gene homologs with the fast-growing m. abscessus possessing paralogous genes. figure shows an alignment of twenty seven kda family proteins identified from genome sequencing projects. displayed as a neighbour-joining tree, it is apparent that the kda proteins fall into three general sub-families: lpqh-like proteins, lppe-like proteins and a third subfamily that we term lp. all except one protein contain a predicted secretion signal sequence with the n-terminus of mature proteins containing a cysteine residue. twenty-one out of twenty-six predicted full-length kda proteins including the m. tuberculosis lpqh and lppe proteins, comply with the lipobox consensus acylation motif. this is consistent with the approximately 75% predictive value of the lipobox based on experimental evidence of known prokaryote lipoproteins. cysteine residues at positions and and phenylalanine at position are conserved throughout the family. strongly and weakly conserved groups of amino acids are also highlighted in figure 2b. o-glycosylation does not occur at a particular motif of amino acids but occurs at specific residues, generally threonine and serine. the m. tuberculosis lpqh kda protein is glycosylated at a triplet and a pair of threonines at positions 14– and 19–. threonine pairs are also found in several other kda family proteins including, for example, the predicted protein from n. farcinica which has two pairs of threonine residues at positions 11– and 15– in addition, many of the kda homologs have n-terminal regions of the mature protein that are rich in serine residues which may be indicative of glycosylation. taken together, it seems likely that n-terminal glycosylation and acylation are general features of the kda protein family. the broad distribution of this family across mycobacteria and closely related genera suggests that these lipoproteins fulfil some conserved physiological function which at present remains largely unknown. to screen for a possible role for the kda lipoprotein in mycobacterial physiology, we therefore generated a deletion mutant lacking the kda molecule and complemented this mutant with the wild type and site-mutagenised copies of the kda molecule. generation and characterization of recombinant m. tuberculosis strains pcr analysis showed rv to be absent from Δ and that this sequence had been successfully reintroduced into strains Δ19::, Δ19::19na, and Δ19::19nog. western blotting of cellular pellet indicated that the kda was not produced in Δ. expression of native protein of the same mw was restored close to normal levels by reintroduction of the kda gene in strain Δ19::. kda protein was only detected in the supernatant of cultures of the non-acylated and non-o-glycosylated complemented strains and was of slightly lower mw than the native kda. in middlebrook 7h broth the growth rate of the Δ Δ19:: Δ19::19na, and Δ19::19nog strains was identical. cytokine secretion human monocytes were infected with equal numbers of bacilli and co-cultured for hours. during this period, the median secretion of il-1β was significantly reduced by deletion of the kda gene. introduction of the native kda gene as Δ19:: restored secretion to wild type levels but the response to Δ19::19na and Δ19::19nog remained significantly less when compared to Δ19::. there was no difference between h37rv, Δ and Δ19:: in their ability to elicit il-12p or tnf from monocytes. although the response to both the Δ19::19na and Δ19::19nog strains tended to be lower, these differences were also not significantly different from h37rv. induction of apoptosis culture supernatants from donors were also assayed for the presence of histone associated dna fragments, a marker of apoptosis. results for each subject were normalised to unstimulated cells to generate an enrichment factor. the Δ and Δ19::19na and Δ19::19nog were associated with lower levels than h37rv or the Δ19:: strain. however responses varied considerably between donors and none of these trends attained statistical significance. discussion we have investigated deletion of the kda lipoprotein from m. tuberculosis and chromosomal complementation of the deletion mutant by the wild type gene and site directed mutagenised variants lacking motifs for acylation and o-glycosylation. we have determined that both acylation and o-glycosylation are necessary for the protein to remain within the cell. consistent with our previous findings, the kda is an important stimulus for the production of pro-inflammatory il-1β, an effect that is dependent on acylation and o-glycosylation. the effect of deletion and complementation on il-12p and tnf secretion was less marked with no statistically significant differences between strains. although deletion of the kda reduced apoptosis, an effect that could also only be reversed by complementation with the wild type gene, the results were variable between donors and did not attain statistical significance. an interesting finding was that kda protein was only detected in the supernatant of cultures of the non-acylated and non-o-glycosylated complemented strains, whereas the Δ19:: strain expressed the molecule in both pellet and supernatant. this suggests that in order to be retained within the cell wall both acylation and glycosylation are necessary for anchoring within the cell wall. whether this relates to a specific physicochemical interaction or merely reflects the recognised hydrophobicity of the mycobacterial cell membrane remains to be determined. sartain and belisle have recently shown that o-glycosylation affects the positioning in the cell wall but not the enzymatic activity of the superoxide dismuase sodc. in a previous study overexpression of the kda in m. smegmatis reduced its capacity to induce the secretion of il-12p and tnf. this effect was dependent on acylation and glycosylation, as tranformation of, m. smegmatis with na and nog variants of the kda did not reduce the secretion of these cytokines. by contrast overexpression of the native kda molecule in Δ strain of virulent m. tuberculosis had precisely the opposite effect, with the production of il-12p and tnf increased irrespective of phagocyte maturity. in this study we reintroduced the kda gene as a single copy into the chromosome of h37rv under the control of its own promoter. we precisely reproduced our previous findings with respect to the effect of deletion of the kda on the cytokine response of monocytes. we have shown that the kda mediated induction of il-1β is dependent on acylation and glycosylation. taken together these and other studies suggest a consistent effect of acylation and o-glycosylation on the cytokine response to the kda, but that the genetic background and level of expression are also important. further evidence in favour of this hypothesis is our recent finding that a naturally occuring m. tuberculosis strain that lacks the kda gene does not have the same in vitro phenotype as the engineered knock out on the rv background. this potentially important finding requires further investigation as much of our knowledge about gene function in m. tuberculosis is inferred from studies of isogenic mutants on the h37rv background. considerable evidence now points to the protective role of macrophage apoptosis in tuberculosis. apoptosis may prevent the release of intracellular components and the spread of mycobacterial infection by sequestering the pathogens within apoptotic bodies. in addition, uptake of apoptotic debris by competent phagocytes allows efficient cross-presentation of m. tuberculosis antigens. thus, the avoidance of apoptosis may be considered a virulence mechanism and a recent study has in fact reported a inverse relationship between the intracellular growth rate and the ability of strains to induce apoptosis. two previous studies have implicated the kda as pro-apoptotic and our results, although variable between donors tend to support this conclusion. however the dependence or otherwise on post-translation modification requires additional work as the findings of lopez et al. suggested that this effect was acylation independent, whereas the trend in our study suggest acylation is necessary.  in conclusion we have presented further evidence of the role of the kda as a key modulator of the human innate immune response. there is considerable evidence that the protein downregulates ifn-γ induced macrophage activation, an effect that will tend to favour bacillary survival during the development of an acquired immune response. on the other hand the molecule will tend to give away the presence of bacilli to the innate system early in infection, perhaps teleologically explaining why it is not upregulated early after infection. in addition, this work provides further evidence of the utility of defined mutants to delineate key determinants of the innate immune response in the context of whole bacilli. authors' contributions kaw, rjw, grs and dby designed the research. rjw, grs and sms derived the recombinant strains using constructs designed and prepared by on and j-lh. kaw and smn performed and interpreted the immunological studies. grs performed the bioinformatic analysis. all authors contributed to analysis and to writing the manuscript. 
the kda lipoprotein of mycobacterium tuberculosis is an important target of the innate immune response. these results confirm in the context of the whole bacillus an important role for post-translational modification of the kda on both the cellular location and immune response to this protein. to investigate the effect of post-translation modification of this protein on innate recognition in the context of the whole bacillus, we derived a recombinant m. tuberculosis h37rv that lacked the kda gene and complemented this strain by reintroduction of the kda gene into the chromosome as a single copy to produce Δ19:: we also reintroduced the kda gene in two modified forms that lacked motifs for acylation and o-glycosylation. the effect of deletion and complementation on il-12p and tnf secretion was less marked with no statistically significant differences between strains. il- beta secretion from human monocytes was significantly reduced by deletion of the kda gene. although deletion of the kda reduced apoptosis, an effect that could also only be reversed by complementation with the wild type gene, the results were variable between donors and did not achieve statistical significance. complementation by the wild type, but not the mutagenised gene reversed this phenotype.
 the kda lipoprotein of mycobacterium tuberculosis is an important target of the innate immune response. to investigate the effect of post-translation modification of this protein on innate recognition in the context of the whole bacillus, we derived a recombinant m. tuberculosis h37rv that lacked the kda gene and complemented this strain by reintroduction of the kda gene into the chromosome as a single copy to produce Δ19:: we also reintroduced the kda gene in two modified forms that lacked motifs for acylation and o-glycosylation.  both acylation and o-glycosylation were necessary for the protein to remain within the cell. il- beta secretion from human monocytes was significantly reduced by deletion of the kda gene. complementation by the wild type, but not the mutagenised gene reversed this phenotype. the effect of deletion and complementation on il-12p and tnf secretion was less marked with no statistically significant differences between strains. although deletion of the kda reduced apoptosis, an effect that could also only be reversed by complementation with the wild type gene, the results were variable between donors and did not achieve statistical significance.  these results confirm in the context of the whole bacillus an important role for post-translational modification of the kda on both the cellular location and immune response to this protein. 