Signature tagged mutagenesis in the functional
Keywords : signature tagged mutagenesis , virulence , gastrointestinal , pathogenesis , pathogen , gut , GI tract 
Introduction
PCR . 
The original tag consisted of a short DNA sequence of 40 bp that was flanked by two invariant arms of 20 bp . 
The region between the variable and invariable region contained a restriction enzyme site that could be used to release the arms from the central regions following amplification and labeling therefore allowing for tag specific probes to be generated . 
The original method to detect the signature tags was by dot blot but over the years there have been many methods for detection of signature tags ( PCR , polymorphic tag-length transposon mutagenesis ) . 
STM was initially developed to identify virulence genes in Salm bs onel nter ca s ro r oth r b ter al s e een usBlaieoisecvaityphimurium but has subsequently ed in many screens ineenacciep.cies as well as in the yeast Saccharomyces cerevisiae , the fungus Cryptococcus neoformans and the parasite Toxoplasma gondii.1-10 stribute . 
For any bacterial pathogen there are several critical parameters that must be followed in order to ensure an efficient STM screen in vivo . 
First , the transposon chosen should insert randomly into the chromosome , a property which varies depending on the transposon . 
It was previously noted that the Tn917 transposon system used in Listeria monocytogenes has tendency for hot-spots , while this is not the case with a recently developed marnier transposon system pJZ037.11-14 Second , the pool size must be determined with respect to the inoculum dose ( normally this ranges between 48 -- 96 mutants per pool ) . 
Finally the route of administration and the infecting dose must be established and the best time frame for when to evaluate a possible attenuation in virulence . 
The main advantages of this system compared with other classical gene inactivation methods ( targeted or random ) is that STM is a negative selection screen allowing the discovery of virulence genes without prior knowledge of their nature or function .15 Secondly , as a large number of mutants can be screened in tandem ( up to 96 ) , this method is in principal much faster and more exhaustive in identifying virulence factors compared with standard transposon systems . 
The disadvantage of this system is that it is limited to finding non-essential genes ( i.e. , genes not required for growth in broth ) . 
Furthermore some DNA tags are unable to be amplified from chromosomal DNA of bacteria after recovery from the animal host . 
This reason for this is not known but it can result in loss of reproducibility and the identification of false negatives . 
This can be overcome by re-organizing the candidates in a new pool that is then re-screened in the animal . 
While this may be time-consuming it reduces the number of false-negative candidates . 
Another drawback encountered with infections , particularly diarrheal disease , are still the third most common cause of death in children under 5 years of age .19 An example of such a disease is Salmonella enterica serovar Typhi infection ( typhoid fever ) , which results in more than 2 million infections a year leading to approximately 200,000 deaths .20 Bacterial pathogens have developed several intricate systems to evade detection by the immune response and to circumnavigate the stresses they may encounter in the GI tract ( pH , osmotic stress , bile and acid stress ) .21 -23 Following ingestion , the first physical stress encountered by the bacterium is the low pH of the stomach ( pH 2 ) , followed by the increased osmolarity of the upper small intestine ( equivalent to 0.3 M NaCl ) and in the duodenum , the antimicrobial activity of the biological detergent bile ( 1 L of bile is produced in the liver , stored interdigestively in the gall bladder and secreted into the duodenum each day ) .24 which functions as a Ca2 + dependent metallo-protease with collagenolytic activity . 
The mutant strain produced half as much proteolytic activity as the wild-type and was unable to degrade collagen .26 Collagen type I and III are important components of the extracellular matrix of the stomach epithelium . 
Furthermore type I collagen is present in the area around gastric ulcers and is important for the process of ulcer healing .27 Kavermann and colleagues suggest that the secretion of a collagen degrading enzyme by H. pylori could be responsible for the persistence of gastric or duodenal ulcerogenesis and the delayed healing process . 
Vibrio cholerae
Cholera is an acute diarrheal disease which is characterized by discharge of voluminous rice water stool caused by toxigenic Vibrio cholerae strains .28 The pathogen enters the host through the oral route of infection , transits the gastric acid barrier of the stomach and colonizes the small intestine . 
Once established within this niche the bacteria begin to produce the cholera toxin ( CT ) , which is responsible for the diarrheal disease that is characteristic of cholera .28 The ensuing diarrheal disease can lead to death by dehydration within hours of infection . 
V. cholerae O1 and O139 serogroups producing cholera toxin ( CT ) are mainly responsible for cholera outbreaks that can cause havoc in highly populated regions in Asia , Africa and Latin America .28 accumulation of intracellular K + . 
However this screen was the first to link reduced colonization and decreased survival in organic acids to a gshB mutation in V. cholerae . 
An additional virulence factor shown to be important in colonization and acid tolerance response was a HepA homolog . 
HepA was originally identified in E. coli as a protein that co-purified with RNA polymerase and inhibited the binding of sigma 70.38 A mutation in E. coli in this gene leads to increased sensitivity to UV damage .38 It is thought that acid stress causes DNA damage and if HepA is required for synthesis of the DNA repair enzymes it would establish a link between the two functions . 
Both of these mutations resulted in a 1000 fold decrease in colonization signifying the importance of both of these genes in the infant mouse model .37 An interesting finding from this screen is that over 20 % of the genes identified in the screen play a role in energy metabolism . 
This indicates that the environment in the small intestine of the suckling mouse is nutrient limited . 
Therefore to survive within such an environment V. cholerae must actively employ multiple pathways for energy source acquisition and survival . 
Es is a o m n em t n s rain ha e t e
. 
coli Bcimoosmcber of the commensal bacteria of the large intestine , however ceriaietnscveh.ability to cause disease . 
Enterohemorrhagic E. coli ( EHEC ) is known to cause disease in humans associated with diarrhea and hemorrhagic colitis.39-41 EHEC serotype O157 : H7 has emerged as a major cause of severe diarrhea worldwide and EHEC is the leading stribute . 
predecessor to pediatric acute renal failure in many countries .42,43 Healthy ruminants are the principal reservoir of EHEC and human infections occur by ingestion of contaminated meat or dairy products contaminated with ruminant feces .44,45 To increase the knowledge of EHEC factors associated with bovine coloni-zation a STM mutant bank was created in O157 : H7 background .46 A total of 1900 mutants were screened by oral inoculation of 10 -- 14-day old calves with recovery of the output pool 5 days post-infection . 
79 mutants were identified to be absent or poorly represented in the output pool . 
All the genes were grouped according to their function and consisted of genes involved in TTSS , surface structure , O-islands , regulatory genes , genes involved in central intermediary metabolism and hypothetical genes . 
Thirteen transposons were inserted into genes on the locus of enterocyte effacement ( LEE ) . 
LEE encodes a TTSS required for formation of attaching and effacing lesions on the intestinal epithelia . 
Their data demonstrated that the structural component of TTSS escC plays a vital role in colonization of the calves as this mutant was highly attenuated following oral infection of calves . 
This was the first time that the structural components of the TTSS were implicated in colonization of the calf intestine .46 Furthermore this work demonstrated that coloni-zation of the bovine intestines requires multiple elements not associated with LEE . 
Their evidence suggests that a novel fimbrial locus ( z2199-z2206 ) plays an important function in intestinal colonization .46 This was the first comprehensive study to elucidate the genes required by E. coli O157 : H7 for infection of bovine intestine and this new information can be used to facilitate the grouped into two classes , with class B pilins being associated with intestinal infections .53 Mundy and colleagues suggest that disruption of cfcH is likely to prevent the assembly and/or secretion of CFC pili .50 This would result in C. rodentium cells being unable to adhere to colonic epithelia and therefore unable to establish an infection .50 Another gene identified in this screen encodes a novel type III secreted protein , EspI , which is encoded outside the LEE region and is present in the sequenced A/E EHEC and EPEC pathogens .49 The function of this gene has not been elucidated but it has been shown to play an important role in both bacterial colonization of colonic epithelium of infected mice and induction of hyperplasia in the colonic epithelium of infected mice .49 The second STM screen performed in C. rodentium utilized the C57BL/6 mouse model . 
The study examined 576 mutants of which 19 were attenuated for survival at 5 -- 7 d post-infection .48 Several insertions corresponded to previously identified virulence genes , including the gene cluster cfc and the espI . 
However one interesting finding was an insertion in the gene encoding a putative translocation effector of A/E pathogens , NleB .48,54 An nleB deletion mutant was constructed and tested for its ability to colonize the mouse . 
As with the transposon mutant the nleB deletion mutant was outcompeted by the wild-type in mixed ins ns . 
I ddit on si g e in ecti ns t w fectioBniaoisincnilefnocieas.also shown to be e sential for colonization and virulence .48 NleB is also present in the EHEC O157 : H7 strain indicating that C. rodentium is an invaluable small animal model to represent other A/E pathogens and to test the role of new effector proteins in disease .48 stribute . 
Campylobacter jejuni
Campylobacter jejuni is the most common bacterial cause of food-borne disease in the developed world , with estimates that it infects 1 out 100 individuals in the United States and United Kingdom .55,56 In the developed world , campylobacteriosis is common in neonates and young adults resulting in mild bloody diarrhea , abdominal cramps and the presence of fecal leukocytes .57,58 Although the vast majority of cases are self-limiting , campylobacter can cause severe post-infection complications , such as bacteraemia and polyneuropathies such as GuillainBarré and Miller-Fisher syndrome .59 C. jejuni usually infects the avian gastrointestinal ( GI ) tract particularly of chickens .60 During the slaughtering process , the GI contents may contaminate the meat products and ingestion and handling of contaminated meats are a main cause of sporadic cases of C. jejuni disease .55 STM has been used in both early and late chicken models of infection with varying degrees of success . 
The first STM screen in C. jejuni analyzed cecal colonization of chicks in a 1-d old chick model of commensalism .61 In total 1550 C. jejuni mutants were screened of which 29 were attenuated for colonization representing 22 different genes required for wild type levels of infection .61 The vast majority of the mutants ( 17 ) exhibited a non-motile phenotype or displayed altered flagellar motility .61 It was previously known that motility of C. jejuni is required for wild-type levels of cecal colonization therefore validating the efficacy of the screen .62,63 Of the remaining mutants two were of particular interest Cj0019c and Cj0020c chosen for proof-in-principle of STM because of its excellent genetic systems and well validated animal models .1,73 The original STM screen identified a novel pathogenicity island , SPI-2 74 . 
SPI-2 was only identified due to its role in disseminated infection . 
SPI-2 encodes a type three secretion system ( TTSS ) which is required for intracellular replication and systemic infection .74 Furthermore , SPI-2 genes have been shown to be involved in the survival of Salmonella within the macrophages and play a role in avoidance of NADPH oxidase-dependent killing.75-77 Recently SPI-2 mutants have been included in live attenuated vaccines in S. typhi and S. typhimurium indicating how STM has lead from proof-of-principle all the way to potential clinical applications .78,79 As stated earlier S. typhimurium can colonize a range of different hosts and STM has been used to try and elucidate both species specific virulence factors and common colonization factors to allow a better understanding of how this bacteria is able to infect such a wide variety of different niches . 
A previous mini-Tn5Km2 STM bank was used to screen mutants for attenuated virulence in both calves and chicks .80 S. typhimurium infection in calves results in enterocolitis followed by systemic infection , while infection of 2-week old chicks results in asymptomatic cecal colonization . 
The STM screen in the calves recovered mutants 3s s af r infe tio fro f om ho ge ize -- 5 dayBteiocsncm the ileal mucosa while in the chicks the mutants were recoveredirenmocned.ceca at 4 d post-infection .80 In total 1045 mutants were screened in both hosts . 
Of the screened mutants 75 were associated with attenuation in the calves , 61 were associated with attenuation in chicks alone stribute . 
and 52 mutants were attenuated in both species .80 A large proportion ( n = 40 ) of the mutated genes were within Salmonella pathogenicity islands ( SPIs 1 -- 5 ) . 
All of the mutants with a transposon insertion in SPI-1 or SP1 -- 2 were deficient for colonization of the calf model but only 3/32 of these SPI mutants resulted in poor colonization of the chick ceca .80 This suggests that S. typhimurium is much less dependent on TTSS-1 and TTSS-2 to colonize the intestines of chicks compared with calves .80 Furthermore SPI-4 was found to be required for coloni-zation of calf ileum but not for cecal colonization of chickens .80 Several genes required for production of LPS were identified by this in vivo screen as being attenuated in both calf and chick colonization models .80 LPS is widely considered to play a role in protecting the bacteria against host defense mechanisms such as bile salts , gastric acidity and phagocytes . 
The precise role of LPS in Salmonella virulence is not yet known but it clearly has a major function in colonization of the intestinal sites of different hosts . 
Of the genes associated with attenuation within the chick model several mutants had transposon insertions in genes required for production of six different fimbriae .80 Fimbriae are used by bacteria to adhere to each other as well as host surfaces , and this data indicates that fimbriae are important for colonization within the intestine of the chicken . 
The same STM mutant library was used to identify novel genes associated with virulence in the intestinal colonization of pigs .81 This screen identified 119 mutants attenuated for virulence in the porcine model of infection . 
Of these 119 mutants , the transposon insertion site of 79 had been identified in the previous screen .80 The remaining 40 transposon mutants were associated appropriate doses to be efficiently used to identify Salmonella mutants with altered fitness in vivo .86 Furthermore it is the first step toward a more complete description of Salmonella genes involved in systemic infection , in particular genes that may have a milder phenotype that are difficult to detect by the older STM methods .86 
Listeria monocytogenes
Listeria monocytogenes is a Gram-positive food-borne pathogen responsible for life-threatening infections in humans and animals . 
It is a facultative intracellular pathogen capable of entering a wide variety of host cells , including epithelial cells , hepatocytes , fibroblasts , endothelial cells and macrophages.91-94 Infection occurs in step-wise manner consisting of entry into the host , lysis of the phagosomal vacuole , multiplication in the cytosol and direct cell to cell spread using actin based motility .95 Each step is dependent on virulence factors which are located in a cluster of genes encoding a regulatory protein ( PrfA ) , a phosphatidylinositol specific phospholipase C ( PlcA ) , the hemolysin listeriolysin A ( LLO ) , a metalloprotease ( Mpl ) , an actin recruiting protein ( ActA ) and a lecithinase ( PlcB ) .95 A second locus encodes two proteins involved with invasion , InlA and InlB . 
Expression os vir e ce g nes are c ntr led y t e p ei f theseBulinoescioeolnbchel.otropic regulator PrfA . 
A STM approach was implemented in the L. monocytogenes EGDe background using a Tn917 derivative transposon . 
Mutants se re n bute . 
wertscreied in vivo for reduced colonization of the spleen and liver at 72 h post-infection .96 From this screen the response regulator VirR was identified . 
VirR ( Virulence Regulator ) has high homology to the OmpR-PhoB family of regulators .96 It is part of a seven gene operon , which also contains the sensor kinase , VirS . 
The VirRS two-component system ( TCS ) is novel in that unlike most TCS the constituent genes are not adjacent to each other but are separated by three other loci .96 The results obtained from this study demonstrated that the DvirR strain had a reduced ability to grow and multiply within both liver and spleen even after 24 h indicating a crucial role for VirR in the establishment of successful L. monocytogenes infection .96 This STM screen also identified another novel virulence factor in L. monocytogenes designated FbpA . 
This gene has strong homology to atypical fibronectin-binding proteins such as PavA of Streptococcus pneumoniae , Fpb54 of S. pyogenes and FbpA of S. gordonii.97-99 Fibronectin is a dimeric glyocoprotein that has a critical role in eukaryotic cellular processes such as adhesion , migration and differentiation .100 However , many bacteria such as S. pneumoniae and Staphylococcus aureus utilize fibronectin to facilitate their internalization into epithelial cells .101 Mutation of fbpA in L. monocytogenes resulted in a 100-fold decrease in bacterial counts in the intestine and liver in orally infected mice when compared with the wild-type strain after 72 h. 100 Furthermore , there was a 10-fold decrease in bacterial counts in the mesenteric lymph between mutant and wild-type but no difference in the spleenic bacterial counts .100 This data indicated that FbpA is involved in the hepatic phases of listeriosis and represents a novel virulence factor . 
scores for 1,645 mutants which represented insertions in 855 different genes . 
This represented 91.1 % of mutants analyzed while the previous STM bank only identified insertion sites in 4.2 % of mutants .107 Furthermore the STM screen in O157 : H7 identified 13 attenuating mutations in LEE genes but sequencing identified 54 insertions in the LEE region which corresponded to 21 different genes .107 Analysis of the STM bank of EHEC O26 : H - demonstrated a role for cytotoxins ( EhxA and PssA ) during pathogenesis but these genes were not identified in the EHEC O157 : H7 screen .46,47 Parallel sequencing revealed that several such mutants were represented in the library and were generally negatively selected in calves .107 A similar massively parallel sequencing approach called INSeq ( insertion sequencing ) was developed independently to analyze transposon insertion sites in the gut commensal organism Bacteroides thetaiotaomicron .108 The authors utilized this approach to analyze genes required for colonization of conventional and germ-free or mono-colonized gnotobiotic mice . 
The work revealed how colonization by Bacteroides is influenced by existing populations in the gut and competition for key nutrients in this environment . 
STM is a powerful genetic tool that allows identification of genes that are important for different facets of pathogenesis and is well suited for analysis of elements required for gut colonizas ribute . 
tiontand localized pathogenesis . 
Recent technical advances in the screening , choice and identification of negative selection screens have broadened its applicability and versatility . 
One such technical advance is the development of a positive STM screen .109 This was used to screen for patho-adaptive Pseudomonas aeruginosa mutants promoting survival in the cystic fibrosis lung .109 This novel approach could be applied to other pathogens that enhance fitness in the host through patho-adaptive mutations and could provide a basis for a more comprehensive understanding of chronic infectious disease .109 Overall the STM tool is an important method for better understanding the behavior of microbes in the gut and other environments and in conjunction with other genome wide techniques ( microarray technology , in vivo expression techno-logy , TraDIS ) can be used to fully understand the multi-faceted nature of bacterial pathogenesis . 
It is expected that the results from these STM screens can be used to help develop vaccines or drugs to prevent additional infections and decrease the economic burden associated with such infections . 
Acknowledgments
Joanne Cummins is supported by funding from the Science Foundation Ireland Research Frontiers Programme ( 08-RFP-Gen1320 ) . 
We acknowledge the support of funding from the Alimentary Pharmabiotic Centre , University College Cork under the Science Foundation Ireland Centres for Science Engineering and Technology ( CSET ) program .