BACKGROUND
internalin a  is a sortase achored, cell wall protein and a critical factor in the pathogenesis of the foodborne gram-positive pathogen listeria monocytogenes.
inla stimulates l. monocytogenes entry into normally non-phagocytic intestinal enterocytes  <cit> .
the protein is  <dig> amino acids  in length and composed of seven distinct domains ; , 1- <dig> aa: a consensus n-terminal signal sequence ; , 35- <dig> aa: forms a cap at the n-terminus tip of mature protein ; , 79- <dig> aa:  <dig> leucine rich repeats  with  <dig> containing  <dig> aa  ; , 415- <dig> aa: an inter-repeat domain ; , 518- <dig> aa: three β-repeat domains, which may serve as a stalk to project the sickle shaped lrr out from the cell surface ; and , 767- <dig> aa: a sortase cleavage site  for peptidoglycan cross linking and  771- <dig> aa: a membrane targeting sequence  <cit> .
domain  containing the lrr's of inla is sufficient to stimulate enterocyte uptake  <cit> .
the enterocyte ligand for inla was identified as e-cadherin   <cit> , which is required by host cells for the formation of tight junctions and to promote cellular polarization, communication and differentiation  <cit> .
the localization of cdh <dig> on the basolateral face of differentiated cells suggested that invasion was a secondary event, occurring after non-specific uptake by m cells  <cit> .
oral infection studies using rats  <cit>  and mice  <cit>  provided support for this hypothesis.
however, oral infections resulted in the invasion of enterocytes in a guinea pig model  <cit> .
human colonic caco- <dig> enterocyte cells are also directly permissive to infection in vitro  <cit> .
these seemingly anomalous results are due to the reduced affinity of murine cdh <dig>  for inla.
the reduced affinity was localized to amino acid  <dig> which is a proline in guinea pig and human cdh <dig>  but in rats and mice a glutamic acid is present  <cit> .
this discovery led to the development and application of a transgenic mouse model expressing both human and murine cdh <dig> within intestinal enterocytes, which conclusively demonstrated the role of inla in the pathogenesis of orally acquired l. monocytogenes  <cit> .
in an elegant study, the site of enterocyte cell extrusion at the tips of intestinal villi was identified as a mechanism for exposing cdh <dig> on the apical surface at multicellular junctions  <cit> .
more recently, a transgenic mouse strain that ubiquitously expresses human e-cadherin has been developed to demonstrate a role for inla  in fetoplacental listeriosis  <cit> .
the crystal structure of inla in complex with hcdh <dig> demonstrated the structural importance of proline  <dig> for the interaction  <cit> .
in silico analysis confirmed that the reduced affinity of inla for mcdh1was essentially due to the steric hindrance imposed by the bulky glutamic acid at aa  <dig>  which therefore could not interact with the hydrophobic pocket  created by the removal of one amino acid from lrr  <dig>  <cit> .
overall the crystal structure identified  <dig> residues of hcdh <dig> that interact with the residues across the lrr region.
structural data and the invasion results from previous research  <cit>  have confirmed the essential nature of the lrr's in the inla::cdh <dig> interaction.
small animal model of listeriosis have a number of significant limitations.
even though rabbits and guinea pigs possess a permissive cdh <dig>  they have recently been shown to be resistant to systemic infection due to a species specificity observed in the inlb/host interaction  <cit> .
inlb is required for efficient hepatocyte/endothelial cell invasion in the mouse model and in certain human cell lines.
a novel approach to address the lack of appropriate animal models focused on the 'murinization' of l. monocytogenes rather than the 'humanization' of mice  <cit> .
rational protein design based on the structural data of the inla/hcdh <dig> complex, identified two mutations in inla  that dramatically increased the affinity for both hcdh <dig> and mcdh <dig>  this allowed the development of a variant of l. monocytogenes egd-e  capable of establishing systemic infections in c57bl/6j mice after oral inoculation  <cit> .
however, the strain also exhibited a 2-fold increase in adhesion and consequently invasion into human cells, suggesting that the alteration in tropism towards mice also could enhance the virulence towards humans.
to address any remaining concerns regarding human virulence of murinized l. monocytogenes, we conducted random mutagenesis of inla combined with surface display on a non-invasive, gram-positive, lactococcus lactis to identify mutations that improve the entry into a colonic murine cell line.
using the ct- <dig> cells as a selection tool, multiple positive mutations in independent clones were identified with an enrichment in the inla/hcdh <dig> interacting residues.
the inla genes from  <dig> l. lactis clones were separately recombined into the inla chromosomal locus in egd-eΔinla generating egd-e a to d. also, a version of egd-inlam  <cit>  was created in order to permit comparison with our newly generated inla mutant strains.
in contrast to the strategy employed by wollert et al.
<cit>  we utilised preferred listeria codons for the mutated 192asn and 369ser and designated the strain; egd-e inlam*.
strains were competed against egd-e inlam* in oral murine competitive index assays  <cit> .
a novel aa mutation was identified which enhanced inla/mchd <dig> interaction compared to egd-e. in agreement with earlier studies  <cit>  the adherence/invasion into caco- <dig> cells and virulence by murine intravenous infection of the codon-optimized egd-e inlam* strain was indistinguishable from egd-e, while egd-e inlam* alone exhibited highly reproducible murine oral infections.
methods
bacterial and cell culture
bacterial strains, plasmids and oligonucleotides are described in table  <dig>  for the routine propagation of l. lactis mg <dig> derivative nz <dig>  cells were grown statically at 30°c in m <dig>  broth containing  <dig> % w/v filter sterilized glucose .
l. monocytogenes were cultivated in bhi  and escherichia coli grown in lb at 37°c with shaking at  <dig> rpm.
for growth on agar, respective broths were solidified with  <dig> %  agar .
for blue/white screening in l. monocytogenes, x-gal  was incorporated into bhi agar at  <dig> μg/ml.
antibiotics were added when required: erythromycin e. coli -  <dig> μg/ml, l. monocytogenes -  <dig> μg/ml and chloramphenicol l. lactis -  <dig> μg/ml.
plasmids were isolated from nz <dig> after overnight growth in  <dig> ml of gm <dig>  to lyse, the pellet was resuspended in  <dig> μl of p <dig> buffer  containing  <dig> μg of lysozyme and incubated for  <dig> min at 37°c.
the lysate was processed as described in the qiaprep spin miniprep kit .
a nisin filtrate for pnisa induction was isolated from the supernatant of an overnight l. lactis culture of nz <dig> , aliquots frozen at -20°c.
for all inla induction experiments, overnight l. lactis nz <dig> cultures  were diluted 1: <dig> in  <dig> ml of fresh gm <dig> and grown to an od <dig> nm of  <dig>  .
the expression of inla was induced with  <dig> μl of nisin and grown for a further hour to an od =  <dig>  .
the murine  and human  colonic epithelial cell lines were routinely cultured at 37°c in 5% co <dig>  media was composed of dmem glutamax, 10% fbs, pen/strep and 1% non essential amino acids with all cell culture media purchased from gibco.
oligonucelotides were purchased from eurofins mwg operon.
a restriction sites are highlighted in bold.
mutated triplets are underlined.
the start codon of inla is in italics.
production of electrocompetent lactococcus lactis
the protocol of holo and nes  <cit>  was adapted for the transformation of l. lactis mg <dig> derivative nz <dig>  a gm <dig> overnight culture of nz <dig> was diluted 1: <dig> into  <dig> ml of gm <dig> containing  <dig> mm sucrose and  <dig> % glycine .
this culture was inoculated into  <dig> ml of fresh gs-gm <dig> and grown overnight.
the  <dig> ml culture was inoculated into  <dig> ml of fresh gs-gm <dig>  grown to od <dig> of  <dig>  and cells were subsequently harvested by centrifugation at  <dig>  × g for  <dig> min at 4°c.
the pellet was resuspended in  <dig> ml of ice cold sgb  glucose - filter sterilized), centrifuged, resuspended in  <dig> ml sgb and left on ice for  <dig> min.
the cells were centrifuged, resuspended in  <dig> ml sgb and left on ice for  <dig> min before a final centrifugation and re-suspension with  <dig> ml sgb.
cells were frozen at -80°c in  <dig> μl aliquots.
to electroporate, cells were thawed on ice, mixed with  <dig> ul of pellet paint  precipitated dna and transferred to a  <dig> mm electroporation cuvette .
cells were pulsed at  <dig> kv/cm,  <dig> Ω and  <dig> μf, regenerated in  <dig> ml gm <dig> containing  <dig> mm cacl2/ <dig> mm mgcl <dig> for  <dig>  h and then plated onto gm <dig> agar containing  <dig> μg/ml chloramphenicol.
an efficiency of  <dig> ×  <dig> cfu/μg was routinely obtained with pnz <dig>
cloning of inla into pnzb
the unique bglii site up stream of the nisa promoter in pnz <dig> was removed by linearization of the vector with bglii and ends blunted with t <dig> dna polymerase.
the vector was religated to generate pnzb.
the inla gene was pcr amplified  as described previously  <cit> , digested with ncoi/psti and ligated into the complementary digested pnzb.
ligations were directly electroporated into nz <dig> as described above and the sequence of the inla gene was verified by dna sequencing.
quikchange mutagenesis in l. lactis
primers for site directed mutagenesis   were designed according to the quikchange sdm manual .
all plasmid template isolated from nz <dig> strains was methylated with dam methylase following manufacturer recommendations .
the pcr thermocycling conditions were conducted as described previously  <cit> .
separate  <dig> μl kod hotstart high fidelity polymerase pcr reactions were preformed with each primer for  <dig> cycles and an extension time of  <dig> min  <dig> sec.
after  <dig> cycles the reactions were combined and continued for an additional  <dig> cycles.
amplimers were column purified  and digested overnight with dpni .
digests were pellet paint precipitated and the half of the digest directly electroporated into nz <dig>  between  <dig> and  <dig> colonies were obtained per transformation.
the protocol was repeated to combine sdm changes.
from the final mutagenized plasmids, bglii/bstxi fragments containing the lrr region of inla were excised and ligated into complementary digested pnzbinlawt.
isolation of cell wall proteins
cell wall proteins were isolated from nisin induced  <dig> ml nz9000+pnzbinlawt culture as described by previously  <cit> , except cells were rendered as protoplasts for  <dig> hr at 30°c without mutanolysin.
blotted proteins were probed with the inla specific monoclonal antibody described by hearty et al  <cit> .
random bank of inla mutants in nz9000
the generation of a randomly mutated inla bank between amino acids  <dig> and  <dig>  of inla was accomplished by error prone pcr with mutazyme ii .
plasmid dna  was used as template in the reaction  and a  <dig>  kb fragment amplified between two naturally occurring restriction sites .
from the mutagenesis reactions, four different mutation rates by varying the amount of template used   <dig> ng   <dig> ng   <dig> ng and   <dig>  ng).
this equates to a sliding scale of increasing mutation frequency.
each amplimer pool was digested with bglii and bstxi and ligated into complementary digested pnz8048binla.
the ligations  were pellet paint precipitated and electroporated into electrocompetent nz <dig> .
for each pool a total of  <dig>  colonies were obtained with plasmid religations accounting for  <dig> % of the total .
the colonies from each mutation frequency were pooled and frozen at -80°c.
from each mutation frequency,  <dig> individual colonies were subjected to plasmid isolation as described above and the mutated region sequenced to access the level of mutagenesis.
ct- <dig> and caco- <dig> invasion assays
overnight cultures of nz <dig> containing pnzb only or pnzbinla derivatives  were induced as described above.
a one ml aliquot was then pelleted at  <dig>  × g for  <dig> min and resuspended in  <dig> ml of dmem.
cells were centrifuged, resuspended in fresh dmem and then diluted to a multiplicity of infection of 25: <dig>  l. monocytogenes cells were grown as described previously prior to invasion  <cit> .
ct- <dig>  <cit>  and caco- <dig> cells were seeded at  <dig> ×  <dig> and  <dig> ×  <dig> cells, respectively and grown for  <dig> days until confluency in  <dig> well plates .
on the day prior to use, antibiotics were removed from the media.
on the day of use, cells were washed twice with dmem to remove fbs.
both cell types were invaded for  <dig> h at 37°c in 5% co <dig>  washed once with dulbecco's pbs  and then overlayed with dmem containing  <dig>  or  <dig> μg/ml  gentamicin for  <dig> h. monolayers were washed a further three times with pbs to remove residual antibiotic and then lysed with  <dig> ml of ice cold sterile water.
bacterial cells were enumerated by serial dilution in pbs and plated on gm <dig> agar containing  <dig> μg/ml chloramphenicol.
the remaining lysate from error prone pcr pools were inoculated into gm <dig> containing  <dig> μg/ml chloramphenicol, grown overnight, stocked at -80°c with the protocol repeated for seven passages through ct- <dig> cells.
egd-e derivatives were plated onto bhi agar.
internalin a chromosomal mutagenesis in l. monocytogenes
a  <dig> kb fragment was pcr amplified  from the appropriate mutated pnz8048binla plasmid, with primer design incorporating the first  <dig> nt upstream of the inla gtg start codon.
the amplimers were digested with ncoi/psti, ligated into complementary digested pori <dig> and transformed into e. coli strain ec10b .
the plasmids pori <dig> and pve <dig> we co-transformed into egd-eΔinla and mutagenesis preformed as described by previously  <cit> .
the reconstruction of the inla locus was identified by colony pcr  with the integrity of the gene confirmed by dna sequencing.
intragastric versus intravenous infections of balb/c mice
for all murine experiments, 6- <dig> week old female balb/c mice  were used.
all experiments were approved by the institutional ethics committee.
tail vein intravenous infections were conducted as described previously  <cit>  with an inoculum comprised of equal numbers of egd-e::pimc3kan and egd-e inlam* ::pimc3ery .
for oral inoculation, overnight cultures were centrifuged , washed twice with pbs and resuspended at  <dig> ×  <dig> cfu/ml in pbs containing  <dig> mg/ml of caco <dig>  a  <dig> μl inoculum was comprised of either a single strain  or a two strain mixture .
mice were intragrastrically gavaged and the progression of infection followed over a three day time course.
for bioluminescent imaging, mice were anesthetized on day  <dig> through to day  <dig> with isoflurane gas and imaged in a xenogen ivis  <dig>  at a binning of  <dig> for  <dig> min.
mice were euthanized with spleen and livers aseptically removed, imaged  and enumerated as previously described  <cit> .
RESULTS
a l. monocytogenes gentamicin protection assay for murine cells
invasion into caco- <dig> cells by l. monocytogenes is dependent on the expression of functional inla  <cit> .
we confirmed that a l. monocytogenes mutant producing inla without the lrr and ir domain  is severely compromised in invasion, while an over expressing inla strain exhibits dramatically enhanced invasion .
to establish an equivalent murine assay for l. monocytogenes we used monolayers of ct- <dig> cells  originally isolated from balb/c mice chemically treated to induce tumor formation  <cit> .
while ct- <dig> cells are not enterocyte like , the results from invasion assays showed that they provide characteristics suitable for use as an invasion model .
the Δinla strain displayed a slight reduction  in invasion compared to egd-e, while over expression of inla resulted in a modest increase in invasion.
we speculate that this is due to a reduced affinity of inla for mcdh <dig>  however we have not assayed for mcdh <dig> production by ct- <dig> cells.
heterologous expression was then employed to distinguish inla from additional virulence determinants on the surface of the l. monocytogenes.
we chose to use the well characterized nisin inducible expression system  <cit>   to produce full length inla on the surface of l. lactis.
the system was chosen because production of functional inla on the cell surface of l. lactis had previously been documented  <cit> .
we compared the entry of l. lactis containing vector only , producing wild type inla  or producing inla containing the ser192asn and tyr369ser, but with different codon usage to the previously described murinized inlam  <cit>   into caco- <dig> and ct- <dig> cells.
the presence of inla on the cell surface was confirmed by western blot analysis .
the level of invasion for l. lactis-pnzb into caco- <dig> cells is similar to that observed for egd-eΔinla .
as l. lactis is non invasive, the surviving bacterial cells probably represent bacteria not killed by the gentamicin treatment rather than internalized cells, as documented previously  <cit> .
a similar level of entry into caco- <dig> cells was observed for l. lactis inlawt and l. lactis inlam*, while entry into ct- <dig> cells was 27- <dig> fold greater for l. lactis inlam* compared to l. lactis inlawt .
in contrast to a previous report  <cit> , we observed an increased invasion into a murine cell line by the l. monocytogenes strain over-expressing inlawt in contrast to the plasmid only control .
a similar trend was observed when the l. monocytogenes inla over-expressing strain and Δinla strain were compared  and was also seen in experiments in the l. lactis background .
these results could be due to the high level of inla expression from the pnis and phelp promoters, amplifying the differences in inla on the surface of l. lactis and l. monocytogenes cells .
we interpret these results as evidence of a specific interaction between inla and a cell surface receptor on ct- <dig> cells which stimulates bacterial cell entry.
to summarise, we have established a gentamicin protection assay, capable of discriminating inla mediated invasion into a murine cell line.
generation and screening of a random bank of inla lrr mutants
to generate diversity within the inla gene we applied error prone pcr to the lrr region .
four separate banks were created containing different levels of mutation frequency, each containing about  <dig>  l. lactis clones.
initial assessment by dna sequencing of ten clones from each bank identified mutations throughout the lrr region with the level of mutation correlating with the concentration of input template dna for the error prone pcr .
to identify positive mutations, pools were invaded through ct- <dig> cells en masse as detailed in figure  <dig>  sequential passages through ct- <dig> cells were required to remove the background functional inla from the pools .
of the four banks only the highest mutation frequency resulted in an initial recovery below that of wild type inla, which suggested that a significant number of clones contained inactivating mutations.
from passage two through six a significant enrichment in positive mutations was observed, with a leveling off at passage seven .
from passage six, eight clones from each bank were sequenced  and assayed individually using both ct- <dig> and caco- <dig> cells .
all clones exhibited enhanced entry into ct- <dig> cells while no apparent differences for cell entry into caco- <dig> cells were observed .
however, no clones were identified which were capable of matching the level of l. lactis inlam* mediated entry into the murine cells.
sequence analysis revealed that  <dig> of the  <dig> clones contained amino acid changes in residues involved in direct interaction with cdh <dig>  of the four banks, only the lowest mutation frequency contained multiple clones with the same mutation , with this single amino acid change also found in one clone from an additional bank .
k301i
g303e
n143k
p159a
q196l
k218m
v224a
g303e
l329q
k301i
k301e
n413y
k301i
s223c
n252y
g303e
t446a
t268i
g303e
t446a
s223c
n252y
s311c
l185f
a270g
q190l
p290q
p290s
l404s
n413y
f150v
l203f
y369f
n381i
s308r
y369s
n381i
s292t
e330v
d199v
s377n
p444s
mutations identified in the bglii/bstxi fragment of pnzbinla  and the invasion increase into ct- <dig> cells versus l. lactis inlawt.
the amino acid mutations identified which involved in the interaction between inlawt and hcdh <dig> are highlighted in bold.
details highlighted in bold and italics are mutations recombined in the chromosome of egd-e.
l. lactis inla site directed mutants with fold invasion increase into ct- <dig> cells vs l. lactis inlawt in brackets: s192n , y <dig> s , s192n+y <dig> s .
below: amino acids in inlawt which interact with hcdh <dig> and amino acid changes identified from error prone pcr screen.
r <dig>  n104: d q*, n <dig>  f150: v, e <dig>  e172: t*, q190: l, s <dig>  r <dig>  d <dig>  i <dig>  t <dig>  e <dig>  n259: y, k301: i e g, n321: y, e <dig>  n325: d, e <dig>  y <dig>  t <dig>  y <dig>  f <dig>  r <dig>  f <dig>  y369: f s, w <dig>  s <dig>  * n <dig> and e <dig> mutations were found from additional screens and sequencing.
characterization of murinized l. monocytogenes: competitive index assays
four inla sequences conferring enhanced invasion into ct- <dig> cells were selected to be re-created in the chromosome of l. monocytogenes egd-e. the mutations constituted two single aa changes for egd-e a  and egd-e b .
while three aa changes were introduced into egd-e c  and egd-e d .
these mutations were chosen based on the frequency of isolation in l. lactis , the ability to attribute the phenotype to an aa change  and the isolation of mutations all confined within one lrr .
a fifth strain was also created based on the lmo-inlam mutation  <cit> , except with listeria optimized codons for 192asn and 369ser, and was used as a positive control .
sequencing confirmed the integrity of the newly introduced mutations, with equivalent levels of inla expressed on the surface of the strains as compared to egd-e .
inlam strain  was compared to the parental egd-e strain for invasion into caco- <dig> and ct- <dig> monolayers.
no differences in invasion  or adherence  were observed to caco- <dig> cells, while the invasion of egd-e inlam* was significantly higher than egd-e into ct- <dig> cells.
we then compared the virulence of egd-e and egd-e inlam* by competitive index  assays via the intravenous   or intragastric   route in balb/c mice.
for i.v.
inoculated mice, no differences in the kinetics of infection were observed for either strain .
this confirms that the two amino acid changes in inlam do not impact on the virulence of egd-e inlam* once the gastrointestinal tract is bypassed.
however, egd-e inlam* was significantly more virulent when infected by the i.g.
route, with higher counts obtained from livers and spleens and a significantly higher ci value  for both day two  and day three .
neither strain was recovered form the liver nor spleen at day one post infection.
subsequent competitive index experiments were conducted by the i.g.
route comparing egd-e inlam* against the strains expressing the inla mutations identified by the ct- <dig> cell screen .
of the four recreated strains, only egd-e a  gave a higher ci than egd-e in the liver  whereas identical values  were obtained for the spleens.
further experimentation will be required to access the contribution of the n259y mutation, and it would be intriguing to see if the recombination of this mutation into egd-e inlam* would further enhance murine pathogenicity.
it is interesting to note that the strain in which inlam  was recreated  did not exhibit enhanced invasion or adhesion to caco- <dig> cells, which is a marker for human virulence, in contrast to the previously published results  <cit> .
to further explore the progression of i.g.
infection, we repeated the balb/c inoculations with either egd-e or egd-e inlam* tagged with a constitutive bioluminescent lux marker and mice were imaged for bioluminescence on each subsequent day  <cit> .
the egd-e inlam* strain exhibited uniform clinical signs of l. monocytogenes infection by day  <dig>  <cit> , while these characteristics were absent from the egd-e group even prior to sacrifice at day  <dig>  consistent with the clinical scores very little light was observed from the egd-e group, while increasing light levels were obtained from the egd-e inlam* group on days  <dig> and  <dig>  with a distinct foci evident in the abdomen in all  <dig> mice by day  <dig> .
upon ex vivo imaging of the livers, a low signal was present in the gall bladder in  <dig> of the  <dig> egd-e infected mice, whereas a much stronger signal was found from the gall bladders of all egd-e inlam*  infected mice, with infection across the liver also observed .
the egd-e inlam* infected gall bladders were also found to be to twice the size of the egd-e group.
further work is necessary to determine the exact extent of gall bladder colonization in these animals relative to hepatocyte infection.
enumeration of the livers and spleens confirmed that the egd-e inlam* strain produced highly reproducible i.g.
infections, with the levels recovered comparable to day three i.v.
infections in the liver .
a much larger degree of variation was observed in the egd-e group, with statistically significant differences in bacterial counts observed between the two strains .
the mechanism of gall bladder colonization is currently unknown  <cit>  and warrants further investigation.
the egd-e inlam* strain is capable of establishing highly reproducible colonization of the gall bladder upon i.g.
inoculation.
this strain will be extremely useful in examining factors required for gastrointestinal transit and gall bladder colonization.
discussion
it is now well established that the murine model of listeriosis is limited by a poor interaction between the bacterial invasion protein inla and its host ligand mcdh <dig>  this is in direct contrast, to the efficient interaction between inla and hcdh <dig>  the discrepancy is due to a glutamate at residue  <dig> in mouse  e-cadherin rendering these host species relatively resistant to infection by the oral route and limiting their use as laboratory models for certain l. monocytogenes-mediated disease processes  <cit> .
recent studies have developed an engineered mouse strain expressing 'humanized' e-cadherin for studies of oral and fetoplacental listeriosis  <cit> .
an alternative approach has utilized structure-based engineering to 'murinize' the bacterial inla protein in order to increase affinity for murine e-cadherin  <cit> .
this approach has provided key insights into the interaction between inla and cdh <dig>  while murinization was highly successful, we reasoned that additional points of contact may also improve the interaction with mcdh <dig>  we therefore developed a system to select random mutations in inla that enhance invasion of murine cells in order to identify novel amino acid interactions and to determine if 'murinization' of the strain can be improved.
l. lactis was used as a surrogate host for this process in order to prevent generation of listeria mutants with increased affinity for human cells.
previous research had shown that heterologous expression of inla from the native pinla promoter in l. lactis could stimulate invasion into cultured human colonic enterocytes and guinea pig enterocytes in an oral infection model  <cit> .
additional properties of l. lactis such as high transformation efficiency  allowed us to generate multiple random libraries of substantial size and enabled the direct transformation of sdm constructs.
also the nisin inducible system enabled a high level of inla expression on the surface of l. lactis in a background with relatively few sortase a anchored proteins.
the ability of l. lactis inlam* to facilitate uptake into murine cells encouraged us to use multiple rounds of en masse enrichment of inla mutant libraries through ct- <dig> cells.
the cumulative results from each passage showed a continued improvement in the invasion efficiency, suggestive of an enrichment of positive clones.
a surprising level of diversity in inla clones was apparent  with  <dig> of the  <dig> clones analyzed exhibiting unique sequences.
only bank iii with the lowest frequency of mutations exhibited a degree of clonality .
this suggests that we have not yet uncovered the full complement of mutations within the banks which confer enhanced invasion capabilities.
directed evolution of the inla gene has the potential to uncover mutations not predicted by a structure-based approach .
with respect to the q190l mutation the glutamine at residue  <dig> found on lrr  <dig> within the hydrophobic pocket, and forms a hydrogen bond to proline  <dig> in hcdh <dig>  the change to leucine may affect the pocket and improve access of glutamic acid  <dig> in mcdh <dig>  of all the single amino acid changes, the n259y mutation exhibited the single greatest invasion increase into ct- <dig> cells.
combining this mutation with either t399i or l <dig> m was shown to reduce or enhance invasion, respectively, with the negative effect of the t399i confirmed by the reduction in invasion efficiency observed when combined with additional positive mutations .
further biochemical studies will be required to identify the role these mutations play to enhance the interaction with mcdh <dig>  the previously identified single aa changes at residues  <dig> and  <dig>  <cit>  each increased invasion ~ <dig> fold, whereas the combined  <dig> +  <dig> mutations increased invasion ~ <dig> fold.
the identical aa change at residue  <dig> was also isolated from our error prone pcr bank.
however, this clone contained additional mutations that resulted in a reduced level of invasion compared to the  <dig> single mutant.
the cdh <dig> interacting amino acids appear to be highly conserved and recalcitrant to change  <cit> .
from a collection of  <dig> inla gene sequences mapped onto the inla crystal structure  <cit> , three naturally occurring inla variants were identified which could potentially mediate an interaction with hcdh <dig>  with one  also identified through the random mutagenesis approach in our study.
however, while all mutants containing this residue had a positive effect on invasion into ct- <dig> cells, the exact contribution of this residue could not be assessed as additional mutations were present in all clones.
further analysis of individual clones from each bank or the application of additional selection is required due to the diversity uncovered .
this diversity and the enhanced invasion of all the clones examined confirms that amino acids additional to the ones previously examined  <cit>  can modulate the affinity for cdh <dig>
despite the analysis of  <dig> clones from our enriched bank of inla variants, we failed to detect mutations that yielded invasion rates comparable to the murinized inla described by wollert and coworkers  <cit> .
in terms of developing usable models of murine listeriosis the approach of 'murinizing' the bacterial strain arguably has a number of benefits over the development of humanized mouse lines.
development of the modified bacterium will permit utilization of this strain in existing mouse lines  and distribution of the murinized strain is relatively straightforward, as is the creation of new mutations in the egd-e inlam* background.
however, the 2-fold enhanced adherence and invasion to human  cells of the l. monocytogenes lmo-inlam  <cit>  could be a potential cause for concern as it is suggestive of a slight enhancement of virulence towards humans.
the procedure used to create that strain required multiple prolonged incubations at 42°c  <cit> .
it has been recently shown that high temperature growth of l. monocytogenes can induce spontaneous mutation, suggesting that high temperature growth should be minimized to avoid the acquisition of secondary mutations  <cit> .
we re-created the inla mutations described by wollert et al.,  <cit>  to create egd-e inlam* using only two temperature shifts to 37°c and six passages under non-selective conditions  <cit> .
another difference between the lmo-inlam and egd-e inlam* strain were the nucleotide changes made to create the mutated amino acids.
in the egd-e inlam* strain the two codons were chosen based on the codon usage from genome analysis, with the most commonly used triplets applied.
in each case usage was 50% higher than the one used in lmo-inlam.
for the asparagine  <dig>  aat compared to the aac codon was chosen .
while for serine  <dig> tct compared to tcg codon was chosen .
the invasion data for lmo-inlam agreed with the biophysical characterization which showed an enhanced interaction for inla with cdh <dig>  <cit>  however as recently shown, synonymous mutations leading to mrna sequence changes can also affect substrate specificity or protein activity  <cit> .
to access the role of codon usage or strain background, competitive index experiments will need to be conducted to directly compare lmo-inlam with egd-e inlam*.
CONCLUSIONS
the research presented here generated random inla variants with enhanced invasion into the ct- <dig> cell line most likely through an increased affinity for mcdh <dig>  novel mutations in inla were readily identified from the random mutagenesis approach and a number  are worthy of further study.
the approach used here indicates that other random or targeted mutagenesis strategies may uncover mutations that further enhance protein-ligand binding.
in particular we suggest that screening approaches such as biopanning  <cit>  using the first extra cellular domain of mcdh <dig> as bait or a site-saturation mutagenesis approach   <cit>  may uncover further potential interactions.
we have demonstrated that the newly created strain, egd-e inlam* does not have an enhanced affinity for human cells  while displaying highly reproducible oral infections in the mouse model.
the use of this murinized l. monocytogenes strain will prove a useful tool in analysing the gastrointestinal phase of listeriosis.
the additional residues identified here as playing a role in inla::cdh <dig> interactions will inform our ongoing efforts to create safer 'murinised' versions of l. monocytogenes which will help us to combat this often fatal pathogen.
authors' contributions
all authors read and approved the final manuscript.
irm devised the study, carried out the experimental work and wrote the manuscript; pgc carried out murine infection work; ch and cgmg devised and guided the study and helped to draft the manuscript.

 internalin a is a sortase achored, cell wall protein and a critical factor in the pathogenesis of the foodborne gram-positive pathogen listeria monocytogenes. inla stimulates l. monocytogenes entry into normally non-phagocytic intestinal enterocytes. the protein is amino acids in length and composed of seven distinct domains;, 1- aa: a consensus n-terminal signal sequence;, 35- aa: forms a cap at the n-terminus tip of mature protein;, 79- aa: leucine rich repeats with containing aa;, 415- aa: an inter-repeat domain;, 518- aa: three β-repeat domains, which may serve as a stalk to project the sickle shaped lrr out from the cell surface; and, 767- aa: a sortase cleavage site for peptidoglycan cross linking and 771- aa: a membrane targeting sequence. domain containing the lrr's of inla is sufficient to stimulate enterocyte uptake. the enterocyte ligand for inla was identified as e-cadherin, which is required by host cells for the formation of tight junctions and to promote cellular polarization, communication and differentiation. the localization of cdh on the basolateral face of differentiated cells suggested that invasion was a secondary event, occurring after non-specific uptake by m cells. oral infection studies using rats and mice provided support for this hypothesis. however, oral infections resulted in the invasion of enterocytes in a guinea pig model. human colonic caco- enterocyte cells are also directly permissive to infection in vitro. these seemingly anomalous results are due to the reduced affinity of murine cdh for inla. the reduced affinity was localized to amino acid which is a proline in guinea pig and human cdh but in rats and mice a glutamic acid is present. this discovery led to the development and application of a transgenic mouse model expressing both human and murine cdh within intestinal enterocytes, which conclusively demonstrated the role of inla in the pathogenesis of orally acquired l. monocytogenes. in an elegant study, the site of enterocyte cell extrusion at the tips of intestinal villi was identified as a mechanism for exposing cdh on the apical surface at multicellular junctions. more recently, a transgenic mouse strain that ubiquitously expresses human e-cadherin has been developed to demonstrate a role for inla in fetoplacental listeriosis. the crystal structure of inla in complex with hcdh demonstrated the structural importance of proline for the interaction. in silico analysis confirmed that the reduced affinity of inla for mcdh1was essentially due to the steric hindrance imposed by the bulky glutamic acid at aa which therefore could not interact with the hydrophobic pocket created by the removal of one amino acid from lrr. overall the crystal structure identified residues of hcdh that interact with the residues across the lrr region. structural data and the invasion results from previous research have confirmed the essential nature of the lrr's in the inla::cdh interaction. small animal model of listeriosis have a number of significant limitations. even though rabbits and guinea pigs possess a permissive cdh they have recently been shown to be resistant to systemic infection due to a species specificity observed in the inlb/host interaction. inlb is required for efficient hepatocyte/endothelial cell invasion in the mouse model and in certain human cell lines. a novel approach to address the lack of appropriate animal models focused on the'murinization' of l. monocytogenes rather than the'humanization' of mice. rational protein design based on the structural data of the inla/hcdh complex, identified two mutations in inla that dramatically increased the affinity for both hcdh and mcdh this allowed the development of a variant of l. monocytogenes egd-e capable of establishing systemic infections in c57bl/6j mice after oral inoculation. however, the strain also exhibited a 2-fold increase in adhesion and consequently invasion into human cells, suggesting that the alteration in tropism towards mice also could enhance the virulence towards humans. to address any remaining concerns regarding human virulence of murinized l. monocytogenes, we conducted random mutagenesis of inla combined with surface display on a non-invasive, gram-positive, lactococcus lactis to identify mutations that improve the entry into a colonic murine cell line. using the ct- cells as a selection tool, multiple positive mutations in independent clones were identified with an enrichment in the inla/hcdh interacting residues. the inla genes from l. lactis clones were separately recombined into the inla chromosomal locus in egd-eΔinla generating egd-e a to d. also, a version of egd-inlam was created in order to permit comparison with our newly generated inla mutant strains. in contrast to the strategy employed by wollert et al. we utilised preferred listeria codons for the mutated 192asn and 369ser and designated the strain; egd-e inlam*. strains were competed against egd-e inlam* in oral murine competitive index assays. a novel aa mutation was identified which enhanced inla/mchd interaction compared to egd-e. in agreement with earlier studies the adherence/invasion into caco- cells and virulence by murine intravenous infection of the codon-optimized egd-e inlam* strain was indistinguishable from egd-e, while egd-e inlam* alone exhibited highly reproducible murine oral infections. methods bacterial and cell culture bacterial strains, plasmids and oligonucleotides are described in table for the routine propagation of l. lactis mg derivative nz cells were grown statically at 30°c in m broth containing % w/v filter sterilized glucose. l. monocytogenes were cultivated in bhi and escherichia coli grown in lb at 37°c with shaking at rpm. for growth on agar, respective broths were solidified with % agar. for blue/white screening in l. monocytogenes, x-gal was incorporated into bhi agar at μg/ml. antibiotics were added when required: erythromycin e. coli - μg/ml, l. monocytogenes - μg/ml and chloramphenicol l. lactis - μg/ml. plasmids were isolated from nz after overnight growth in ml of gm to lyse, the pellet was resuspended in μl of p buffer containing μg of lysozyme and incubated for min at 37°c. the lysate was processed as described in the qiaprep spin miniprep kit. a nisin filtrate for pnisa induction was isolated from the supernatant of an overnight l. lactis culture of nz, aliquots frozen at -20°c. for all inla induction experiments, overnight l. lactis nz cultures were diluted 1: in ml of fresh gm and grown to an od nm of. the expression of inla was induced with μl of nisin and grown for a further hour to an od =. the murine and human colonic epithelial cell lines were routinely cultured at 37°c in 5% co media was composed of dmem glutamax, 10% fbs, pen/strep and 1% non essential amino acids with all cell culture media purchased from gibco. oligonucelotides were purchased from eurofins mwg operon. a restriction sites are highlighted in bold. mutated triplets are underlined. the start codon of inla is in italics. production of electrocompetent lactococcus lactis the protocol of holo and nes was adapted for the transformation of l. lactis mg derivative nz a gm overnight culture of nz was diluted 1: into ml of gm containing mm sucrose and % glycine. this culture was inoculated into ml of fresh gs-gm and grown overnight. the ml culture was inoculated into ml of fresh gs-gm grown to od of and cells were subsequently harvested by centrifugation at × g for min at 4°c. the pellet was resuspended in ml of ice cold sgb glucose - filter sterilized), centrifuged, resuspended in ml sgb and left on ice for min. the cells were centrifuged, resuspended in ml sgb and left on ice for min before a final centrifugation and re-suspension with ml sgb. cells were frozen at -80°c in μl aliquots. to electroporate, cells were thawed on ice, mixed with ul of pellet paint precipitated dna and transferred to a mm electroporation cuvette. cells were pulsed at kv/cm, Ω and μf, regenerated in ml gm containing mm cacl2/ mm mgcl for h and then plated onto gm agar containing μg/ml chloramphenicol. an efficiency of × cfu/μg was routinely obtained with pnz cloning of inla into pnzb the unique bglii site up stream of the nisa promoter in pnz was removed by linearization of the vector with bglii and ends blunted with t dna polymerase. the vector was religated to generate pnzb. the inla gene was pcr amplified as described previously, digested with ncoi/psti and ligated into the complementary digested pnzb. ligations were directly electroporated into nz as described above and the sequence of the inla gene was verified by dna sequencing. quikchange mutagenesis in l. lactis primers for site directed mutagenesis were designed according to the quikchange sdm manual. all plasmid template isolated from nz strains was methylated with dam methylase following manufacturer recommendations. the pcr thermocycling conditions were conducted as described previously. separate μl kod hotstart high fidelity polymerase pcr reactions were preformed with each primer for cycles and an extension time of min sec. after cycles the reactions were combined and continued for an additional cycles. amplimers were column purified and digested overnight with dpni. digests were pellet paint precipitated and the half of the digest directly electroporated into nz between and colonies were obtained per transformation. the protocol was repeated to combine sdm changes. from the final mutagenized plasmids, bglii/bstxi fragments containing the lrr region of inla were excised and ligated into complementary digested pnzbinlawt. isolation of cell wall proteins cell wall proteins were isolated from nisin induced ml nz9000+pnzbinlawt culture as described by previously, except cells were rendered as protoplasts for hr at 30°c without mutanolysin. blotted proteins were probed with the inla specific monoclonal antibody described by hearty et al. random bank of inla mutants in nz9000 the generation of a randomly mutated inla bank between amino acids and of inla was accomplished by error prone pcr with mutazyme ii. plasmid dna was used as template in the reaction and a kb fragment amplified between two naturally occurring restriction sites. from the mutagenesis reactions, four different mutation rates by varying the amount of template used ng ng ng and ng). this equates to a sliding scale of increasing mutation frequency. each amplimer pool was digested with bglii and bstxi and ligated into complementary digested pnz8048binla. the ligations were pellet paint precipitated and electroporated into electrocompetent nz. for each pool a total of colonies were obtained with plasmid religations accounting for % of the total. the colonies from each mutation frequency were pooled and frozen at -80°c. from each mutation frequency, individual colonies were subjected to plasmid isolation as described above and the mutated region sequenced to access the level of mutagenesis. ct- and caco- invasion assays overnight cultures of nz containing pnzb only or pnzbinla derivatives were induced as described above. a one ml aliquot was then pelleted at × g for min and resuspended in ml of dmem. cells were centrifuged, resuspended in fresh dmem and then diluted to a multiplicity of infection of 25: l. monocytogenes cells were grown as described previously prior to invasion. ct- and caco- cells were seeded at × and × cells, respectively and grown for days until confluency in well plates. on the day prior to use, antibiotics were removed from the media. on the day of use, cells were washed twice with dmem to remove fbs. both cell types were invaded for h at 37°c in 5% co washed once with dulbecco's pbs and then overlayed with dmem containing or μg/ml gentamicin for h. monolayers were washed a further three times with pbs to remove residual antibiotic and then lysed with ml of ice cold sterile water. bacterial cells were enumerated by serial dilution in pbs and plated on gm agar containing μg/ml chloramphenicol. the remaining lysate from error prone pcr pools were inoculated into gm containing μg/ml chloramphenicol, grown overnight, stocked at -80°c with the protocol repeated for seven passages through ct- cells. egd-e derivatives were plated onto bhi agar. internalin a chromosomal mutagenesis in l. monocytogenes a kb fragment was pcr amplified from the appropriate mutated pnz8048binla plasmid, with primer design incorporating the first nt upstream of the inla gtg start codon. the amplimers were digested with ncoi/psti, ligated into complementary digested pori and transformed into e. coli strain ec10b. the plasmids pori and pve we co-transformed into egd-eΔinla and mutagenesis preformed as described by previously. the reconstruction of the inla locus was identified by colony pcr with the integrity of the gene confirmed by dna sequencing. intragastric versus intravenous infections of balb/c mice for all murine experiments, 6- week old female balb/c mice were used. all experiments were approved by the institutional ethics committee. tail vein intravenous infections were conducted as described previously with an inoculum comprised of equal numbers of egd-e::pimc3kan and egd-e inlam*::pimc3ery. for oral inoculation, overnight cultures were centrifuged, washed twice with pbs and resuspended at × cfu/ml in pbs containing mg/ml of caco a μl inoculum was comprised of either a single strain or a two strain mixture. mice were intragrastrically gavaged and the progression of infection followed over a three day time course. for bioluminescent imaging, mice were anesthetized on day through to day with isoflurane gas and imaged in a xenogen ivis at a binning of for min. mice were euthanized with spleen and livers aseptically removed, imaged and enumerated as previously described.  a l. monocytogenes gentamicin protection assay for murine cells invasion into caco- cells by l. monocytogenes is dependent on the expression of functional inla. we confirmed that a l. monocytogenes mutant producing inla without the lrr and ir domain is severely compromised in invasion, while an over expressing inla strain exhibits dramatically enhanced invasion. to establish an equivalent murine assay for l. monocytogenes we used monolayers of ct- cells originally isolated from balb/c mice chemically treated to induce tumor formation. while ct- cells are not enterocyte like, the results from invasion assays showed that they provide characteristics suitable for use as an invasion model. the Δinla strain displayed a slight reduction in invasion compared to egd-e, while over expression of inla resulted in a modest increase in invasion. we speculate that this is due to a reduced affinity of inla for mcdh however we have not assayed for mcdh production by ct- cells. heterologous expression was then employed to distinguish inla from additional virulence determinants on the surface of the l. monocytogenes. we chose to use the well characterized nisin inducible expression system to produce full length inla on the surface of l. lactis. the system was chosen because production of functional inla on the cell surface of l. lactis had previously been documented. we compared the entry of l. lactis containing vector only, producing wild type inla or producing inla containing the ser192asn and tyr369ser, but with different codon usage to the previously described murinized inlam into caco- and ct- cells. the presence of inla on the cell surface was confirmed by western blot analysis. the level of invasion for l. lactis-pnzb into caco- cells is similar to that observed for egd-eΔinla. as l. lactis is non invasive, the surviving bacterial cells probably represent bacteria not killed by the gentamicin treatment rather than internalized cells, as documented previously. a similar level of entry into caco- cells was observed for l. lactis inlawt and l. lactis inlam*, while entry into ct- cells was 27- fold greater for l. lactis inlam* compared to l. lactis inlawt. in contrast to a previous report, we observed an increased invasion into a murine cell line by the l. monocytogenes strain over-expressing inlawt in contrast to the plasmid only control. a similar trend was observed when the l. monocytogenes inla over-expressing strain and Δinla strain were compared and was also seen in experiments in the l. lactis background. these results could be due to the high level of inla expression from the pnis and phelp promoters, amplifying the differences in inla on the surface of l. lactis and l. monocytogenes cells. we interpret these results as evidence of a specific interaction between inla and a cell surface receptor on ct- cells which stimulates bacterial cell entry. to summarise, we have established a gentamicin protection assay, capable of discriminating inla mediated invasion into a murine cell line. generation and screening of a random bank of inla lrr mutants to generate diversity within the inla gene we applied error prone pcr to the lrr region. four separate banks were created containing different levels of mutation frequency, each containing about l. lactis clones. initial assessment by dna sequencing of ten clones from each bank identified mutations throughout the lrr region with the level of mutation correlating with the concentration of input template dna for the error prone pcr. to identify positive mutations, pools were invaded through ct- cells en masse as detailed in figure sequential passages through ct- cells were required to remove the background functional inla from the pools. of the four banks only the highest mutation frequency resulted in an initial recovery below that of wild type inla, which suggested that a significant number of clones contained inactivating mutations. from passage two through six a significant enrichment in positive mutations was observed, with a leveling off at passage seven. from passage six, eight clones from each bank were sequenced and assayed individually using both ct- and caco- cells. all clones exhibited enhanced entry into ct- cells while no apparent differences for cell entry into caco- cells were observed. however, no clones were identified which were capable of matching the level of l. lactis inlam* mediated entry into the murine cells. sequence analysis revealed that of the clones contained amino acid changes in residues involved in direct interaction with cdh of the four banks, only the lowest mutation frequency contained multiple clones with the same mutation, with this single amino acid change also found in one clone from an additional bank. k301i g303e n143k p159a q196l k218m v224a g303e l329q k301i k301e n413y k301i s223c n252y g303e t446a t268i g303e t446a s223c n252y s311c l185f a270g q190l p290q p290s l404s n413y f150v l203f y369f n381i s308r y369s n381i s292t e330v d199v s377n p444s mutations identified in the bglii/bstxi fragment of pnzbinla and the invasion increase into ct- cells versus l. lactis inlawt. the amino acid mutations identified which involved in the interaction between inlawt and hcdh are highlighted in bold. details highlighted in bold and italics are mutations recombined in the chromosome of egd-e. l. lactis inla site directed mutants with fold invasion increase into ct- cells vs l. lactis inlawt in brackets: s192n, y s, s192n+y s. below: amino acids in inlawt which interact with hcdh and amino acid changes identified from error prone pcr screen. r n104: d q*, n f150: v, e e172: t*, q190: l, s r d i t e n259: y, k301: i e g, n321: y, e n325: d, e y t y f r f y369: f s, w s * n and e mutations were found from additional screens and sequencing. characterization of murinized l. monocytogenes: competitive index assays four inla sequences conferring enhanced invasion into ct- cells were selected to be re-created in the chromosome of l. monocytogenes egd-e. the mutations constituted two single aa changes for egd-e a and egd-e b. while three aa changes were introduced into egd-e c and egd-e d. these mutations were chosen based on the frequency of isolation in l. lactis, the ability to attribute the phenotype to an aa change and the isolation of mutations all confined within one lrr. a fifth strain was also created based on the lmo-inlam mutation, except with listeria optimized codons for 192asn and 369ser, and was used as a positive control. sequencing confirmed the integrity of the newly introduced mutations, with equivalent levels of inla expressed on the surface of the strains as compared to egd-e. inlam strain was compared to the parental egd-e strain for invasion into caco- and ct- monolayers. no differences in invasion or adherence were observed to caco- cells, while the invasion of egd-e inlam* was significantly higher than egd-e into ct- cells. we then compared the virulence of egd-e and egd-e inlam* by competitive index assays via the intravenous or intragastric route in balb/c mice. for i.v. inoculated mice, no differences in the kinetics of infection were observed for either strain. this confirms that the two amino acid changes in inlam do not impact on the virulence of egd-e inlam* once the gastrointestinal tract is bypassed. however, egd-e inlam* was significantly more virulent when infected by the i.g. route, with higher counts obtained from livers and spleens and a significantly higher ci value for both day two and day three. neither strain was recovered form the liver nor spleen at day one post infection. subsequent competitive index experiments were conducted by the i.g. route comparing egd-e inlam* against the strains expressing the inla mutations identified by the ct- cell screen. of the four recreated strains, only egd-e a gave a higher ci than egd-e in the liver whereas identical values were obtained for the spleens. further experimentation will be required to access the contribution of the n259y mutation, and it would be intriguing to see if the recombination of this mutation into egd-e inlam* would further enhance murine pathogenicity. it is interesting to note that the strain in which inlam was recreated did not exhibit enhanced invasion or adhesion to caco- cells, which is a marker for human virulence, in contrast to the previously published results. to further explore the progression of i.g. infection, we repeated the balb/c inoculations with either egd-e or egd-e inlam* tagged with a constitutive bioluminescent lux marker and mice were imaged for bioluminescence on each subsequent day. the egd-e inlam* strain exhibited uniform clinical signs of l. monocytogenes infection by day, while these characteristics were absent from the egd-e group even prior to sacrifice at day consistent with the clinical scores very little light was observed from the egd-e group, while increasing light levels were obtained from the egd-e inlam* group on days and with a distinct foci evident in the abdomen in all mice by day. upon ex vivo imaging of the livers, a low signal was present in the gall bladder in of the egd-e infected mice, whereas a much stronger signal was found from the gall bladders of all egd-e inlam* infected mice, with infection across the liver also observed. the egd-e inlam* infected gall bladders were also found to be to twice the size of the egd-e group. further work is necessary to determine the exact extent of gall bladder colonization in these animals relative to hepatocyte infection. enumeration of the livers and spleens confirmed that the egd-e inlam* strain produced highly reproducible i.g. infections, with the levels recovered comparable to day three i.v. infections in the liver. a much larger degree of variation was observed in the egd-e group, with statistically significant differences in bacterial counts observed between the two strains. the mechanism of gall bladder colonization is currently unknown and warrants further investigation. the egd-e inlam* strain is capable of establishing highly reproducible colonization of the gall bladder upon i.g. inoculation. this strain will be extremely useful in examining factors required for gastrointestinal transit and gall bladder colonization. discussion it is now well established that the murine model of listeriosis is limited by a poor interaction between the bacterial invasion protein inla and its host ligand mcdh this is in direct contrast, to the efficient interaction between inla and hcdh the discrepancy is due to a glutamate at residue in mouse e-cadherin rendering these host species relatively resistant to infection by the oral route and limiting their use as laboratory models for certain l. monocytogenes-mediated disease processes. recent studies have developed an engineered mouse strain expressing'humanized' e-cadherin for studies of oral and fetoplacental listeriosis. an alternative approach has utilized structure-based engineering to'murinize' the bacterial inla protein in order to increase affinity for murine e-cadherin. this approach has provided key insights into the interaction between inla and cdh while murinization was highly successful, we reasoned that additional points of contact may also improve the interaction with mcdh we therefore developed a system to select random mutations in inla that enhance invasion of murine cells in order to identify novel amino acid interactions and to determine if'murinization' of the strain can be improved. l. lactis was used as a surrogate host for this process in order to prevent generation of listeria mutants with increased affinity for human cells. previous research had shown that heterologous expression of inla from the native pinla promoter in l. lactis could stimulate invasion into cultured human colonic enterocytes and guinea pig enterocytes in an oral infection model. additional properties of l. lactis such as high transformation efficiency allowed us to generate multiple random libraries of substantial size and enabled the direct transformation of sdm constructs. also the nisin inducible system enabled a high level of inla expression on the surface of l. lactis in a background with relatively few sortase a anchored proteins. the ability of l. lactis inlam* to facilitate uptake into murine cells encouraged us to use multiple rounds of en masse enrichment of inla mutant libraries through ct- cells. the cumulative results from each passage showed a continued improvement in the invasion efficiency, suggestive of an enrichment of positive clones. a surprising level of diversity in inla clones was apparent with of the clones analyzed exhibiting unique sequences. only bank iii with the lowest frequency of mutations exhibited a degree of clonality. this suggests that we have not yet uncovered the full complement of mutations within the banks which confer enhanced invasion capabilities. directed evolution of the inla gene has the potential to uncover mutations not predicted by a structure-based approach. with respect to the q190l mutation the glutamine at residue found on lrr within the hydrophobic pocket, and forms a hydrogen bond to proline in hcdh the change to leucine may affect the pocket and improve access of glutamic acid in mcdh of all the single amino acid changes, the n259y mutation exhibited the single greatest invasion increase into ct- cells. combining this mutation with either t399i or l m was shown to reduce or enhance invasion, respectively, with the negative effect of the t399i confirmed by the reduction in invasion efficiency observed when combined with additional positive mutations. further biochemical studies will be required to identify the role these mutations play to enhance the interaction with mcdh the previously identified single aa changes at residues and each increased invasion ~ fold, whereas the combined + mutations increased invasion ~ fold. the identical aa change at residue was also isolated from our error prone pcr bank. however, this clone contained additional mutations that resulted in a reduced level of invasion compared to the single mutant. the cdh interacting amino acids appear to be highly conserved and recalcitrant to change. from a collection of inla gene sequences mapped onto the inla crystal structure, three naturally occurring inla variants were identified which could potentially mediate an interaction with hcdh with one also identified through the random mutagenesis approach in our study. however, while all mutants containing this residue had a positive effect on invasion into ct- cells, the exact contribution of this residue could not be assessed as additional mutations were present in all clones. further analysis of individual clones from each bank or the application of additional selection is required due to the diversity uncovered. this diversity and the enhanced invasion of all the clones examined confirms that amino acids additional to the ones previously examined can modulate the affinity for cdh despite the analysis of clones from our enriched bank of inla variants, we failed to detect mutations that yielded invasion rates comparable to the murinized inla described by wollert and coworkers. in terms of developing usable models of murine listeriosis the approach of'murinizing' the bacterial strain arguably has a number of benefits over the development of humanized mouse lines. development of the modified bacterium will permit utilization of this strain in existing mouse lines and distribution of the murinized strain is relatively straightforward, as is the creation of new mutations in the egd-e inlam* background. however, the 2-fold enhanced adherence and invasion to human cells of the l. monocytogenes lmo-inlam could be a potential cause for concern as it is suggestive of a slight enhancement of virulence towards humans. the procedure used to create that strain required multiple prolonged incubations at 42°c. it has been recently shown that high temperature growth of l. monocytogenes can induce spontaneous mutation, suggesting that high temperature growth should be minimized to avoid the acquisition of secondary mutations. we re-created the inla mutations described by wollert et al., to create egd-e inlam* using only two temperature shifts to 37°c and six passages under non-selective conditions. another difference between the lmo-inlam and egd-e inlam* strain were the nucleotide changes made to create the mutated amino acids. in the egd-e inlam* strain the two codons were chosen based on the codon usage from genome analysis, with the most commonly used triplets applied. in each case usage was 50% higher than the one used in lmo-inlam. for the asparagine aat compared to the aac codon was chosen. while for serine tct compared to tcg codon was chosen. the invasion data for lmo-inlam agreed with the biophysical characterization which showed an enhanced interaction for inla with cdh however as recently shown, synonymous mutations leading to mrna sequence changes can also affect substrate specificity or protein activity. to access the role of codon usage or strain background, competitive index experiments will need to be conducted to directly compare lmo-inlam with egd-e inlam*.  the research presented here generated random inla variants with enhanced invasion into the ct- cell line most likely through an increased affinity for mcdh novel mutations in inla were readily identified from the random mutagenesis approach and a number are worthy of further study. the approach used here indicates that other random or targeted mutagenesis strategies may uncover mutations that further enhance protein-ligand binding. in particular we suggest that screening approaches such as biopanning using the first extra cellular domain of mcdh as bait or a site-saturation mutagenesis approach may uncover further potential interactions. we have demonstrated that the newly created strain, egd-e inlam* does not have an enhanced affinity for human cells while displaying highly reproducible oral infections in the mouse model. the use of this murinized l. monocytogenes strain will prove a useful tool in analysing the gastrointestinal phase of listeriosis. the additional residues identified here as playing a role in inla::cdh interactions will inform our ongoing efforts to create safer'murinised' versions of l. monocytogenes which will help us to combat this often fatal pathogen. authors' contributions all authors read and approved the final manuscript. irm devised the study, carried out the experimental work and wrote the manuscript; pgc carried out murine infection work; ch and cgmg devised and guided the study and helped to draft the manuscript. 
the interaction of inla with the host cell ligand e-cadherin efficiently stimulates l. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells. the strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates listeria-optimized codons encoding the altered amino acids. competitive index experiments were conducted in mice using selected mutations introduced into l. monocytogenes egd-e background. this murinized l. monocytogenes strain will provide a useful tool for the analysis of the gastrointestinal phase of listeriosis. after sequential passage through a murine cell line, multiple clones with enhanced invasion characteristics were identified. internalin a is a critical virulence factor which mediates the initiation of listeria monocytogenes infection by the oral route in permissive hosts. a novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. l. monocytogenes egd-e inlam* yielded consistently higher level murine infections by the oral route when compared to egd-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the egd-inlam strain. during the course of the study we engineered a murinized strain of l. monocytogenes egd-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human cells as previously observed. as a control a previously described egd-inlam murinized strain was also re-created as part of this study with minor modifications and designated egd-e inlam*.
 internalin a is a critical virulence factor which mediates the initiation of listeria monocytogenes infection by the oral route in permissive hosts. the interaction of inla with the host cell ligand e-cadherin efficiently stimulates l. monocytogenes entry into human enterocytes, but has only a limited interaction with murine cells.  we have created a surface display library of randomly mutated inla in a non-invasive heterologous host lactococcus lactis in order to create and screen novel variants of this invasion factor. after sequential passage through a murine cell line, multiple clones with enhanced invasion characteristics were identified. competitive index experiments were conducted in mice using selected mutations introduced into l. monocytogenes egd-e background. a novel single amino acid change was identified which enhanced virulence by the oral route in the murine model and will form the basis of further engineering approaches. as a control a previously described egd-inlam murinized strain was also re-created as part of this study with minor modifications and designated egd-e inlam*. the strain was created using a procedure that minimizes the likelihood of secondary mutations and incorporates listeria-optimized codons encoding the altered amino acids. l. monocytogenes egd-e inlam* yielded consistently higher level murine infections by the oral route when compared to egd-e, but did not display the two-fold increased invasion into a human cell line that was previously described for the egd-inlam strain.  we have used both site-directed mutagenesis and directed evolution to create variants of inla which may inform future structure-function analyses of this protein. during the course of the study we engineered a murinized strain of l. monocytogenes egd-e which shows reproducibly higher infectivity in the intragastric murine infection model than the wild type, but does not display enhanced entry into human cells as previously observed. this murinized l. monocytogenes strain will provide a useful tool for the analysis of the gastrointestinal phase of listeriosis. 