Targeted and Random Mutagenesis of Ehrlichia chaffeensis for the Identification of Genes Required for In vivo Infection
[['Ehrlichia chaffeensis , an alpha-proteobacterium , is an intracellular pathogen that is transmitted through an infected Lone Star tick , Amblyomma americanum , to humans and several other vertebrate hosts [1]–[8] .', 'The pathogen is responsible for causing human monocytic ehrlichiosis ( HME ) [6] , [7] , [9] , [10] .', 'The disease is characterized by an acute onset of febrile illness that can progress to a fatal outcome , particularly in immune compromised individuals [2] , [11] .', 'Clinical symptoms of the flu like illness include malaise , nausea , headache , myalgia and persistent fever .', 'Leukopenia , thrombocytopenia , and elevated liver transaminases are common laboratory findings [9] , [10] , [12] .', 'E . chaffeensis and related pathogens have evolved unique strategies to establish infections in both ticks and mammals in order to successfully complete their transmission cycle [13] , [14] .', 'Persistent infection throughout the developmental stages of ticks is important , as the organism cannot be transmitted transovarially to larval offspring .', 'Our recent molecular and proteomic studies have revealed global differences in the expressed proteins of E . chaffeensis within different host cell environments [15]–[18] .', "The pathogen's differential gene expression in response to distinct cellular environments is a major contributor for its dual host adaptation and persistence [19] .", 'Targeted or random mutagenesis is routinely employed to study gene function in bacteria that can be grown axenically .', 'Transformation of obligate intracellular pathogens , such as E . chaffeensis , remains a challenge , particularly considering its limited viability in the extra-cellular environment [20] .', 'Transformation experiments , therefore , must be conducted during a small window of time when the bacteria must remain competent to infect host cells .', "Secondly , as Ehrlichia species do not harbor plasmids , the introduced foreign DNA in the form of a plasmid or linear fragments must remain intact during the transformation and infection stages so that the encoded genes can be expressed using the bacterium's RNA polymerase complex .", 'Finally , the choice of an antibiotic resistance cassette to be introduced into the organisms should not target antibiotics useful in treating a patient .', 'In this study , we considered all these aspects in creating mutational methods in E . chaffeensis .', 'Homologous recombination is useful for targeting mutations to a specific genomic site , and has been successfully employed in making gene disruption mutations in several intracellular bacteria [21]–[24] .', 'Similarly , a modified mobile group II intron ( TargeTron ) -based targeted mutagenesis has been proven valuable for efficient gene targeting in several Gram-negative and Gram-positive bacteria [25]–[31] .', 'Recent studies have shown that the Himar1 transposase system is a valuable tool for transposon-mutagenesis of various bacterial organisms , including alpha-proteobacteria closely related to E . chaffeensis , such as Anaplasma phagocytophilum [32] , [33] .', 'In this study , we evaluated methods for creating targeted mutations in E . chaffeensis by employing both homologous recombination and TargeTron methods , and random mariner mutagenesis using the Himar1 transposase system .', 'Six different genomic locations were assessed by targeted mutagenesis which led to the consistent identification of mutants at three genomic sites .', 'We generated nine random transposon-mediated mutations in the E . chaffeensis genome , three of which disrupted the coding regions of different transcriptionally active hypothetical protein genes , and six in intergenic sites .', 'Four of the insertions also caused loss of gene expression .', 'We present the first evidence that insertion mutations at three sites within the E . chaffeensis genome abolished the growth of the organism in its natural host .'], ['We evaluated the ability of spectinomycin , rifampin , chloramphenicol , gentamicin , and kanamycin and ampicillin to inhibit the growth of E . chaffeensis in the canine macrophage cell line , DH82 .', 'With the exception of ampicillin , all of the antibiotics inhibited the growth of the organism .', 'The minimum concentrations of antibiotics required for 100% growth inhibition by day 7 varied , and they were 100 µg/ml for spectinomycin , 0 . 1 µg/ml for rifampin , 80 µg/ml for gentamicin , 4 µg/ml for chloramphenicol and 50 µg/ml for kanamycin .', 'We opted to use antibiotic resistance gene cassettes against spectinomycin ( which also confers resistance to streptomycin [32] and chloramphenicol for assessing the growth of E . chaffeensis following the introduction of insertion mutations in the organism , as they were proven valuable in similar studies in other intracellular bacteria of the genera Anaplasma , Coxiella and Rickettsia [32] , [34] , [35] .', 'A gentamicin resistance cassette was also used in this study , but as the results for this cassette were similar to the chloramphenicol acetyl transferase ( CAT ) gene cassette ( described below ) , the use of this cassette was not described .', 'For creating targeted insertion mutations , several genomic regions spanning both intergenic and intragenic regions were selected .', 'The genomic targets within protein coding sequences included genes Ech_0126 ( a hypothetical protein gene ) , Ech_1136 ( p28-Omp 14 gene ) and Ech_1143 ( p28-Omp 19 gene ) .', 'The intergenic regions included the DNA segments spanning the genes Ech_0039 and Ech_0040 , Ech_0111 and Ech_0112 , and Ech_0251 and Ech_0252 .', 'The hypothetical protein genes were selected because our RNA analysis ( not shown ) suggested that the bacterium did not transcribe from these genomic regions when propagated in DH82 macrophages .', 'Ech_1136 is a major transcriptionally active gene encoding the p28-Omp 14 protein when the pathogen is grown in tick cells , whereas Ech_1143 gene is highly active in producing the p28-Omp 19 protein during its replication in vertebrate macrophages [15]–[18] .', 'The underlying hypothesis for selecting these two differentially expressed genes is that disruption of a gene expressed exclusively in tick cells should permit normal growth of the organism in macrophages .', 'Similarly , insertion mutations of genes expressed in macrophage environment should allow the pathogen to grow normally in tick cells .', 'Two types of recombinant DNA segments were prepared targeting the gene Ech_0126; in the first version ( Rec I ) , a segment was prepared to introduce an antibiotic resistance gene to disrupt the gene .', 'The recombination event results in the increase of a genome size by 0 . 93 kb .', 'As the increase in genome size may cause polar effects influencing expression of genes surrounding the insertion site , a second type of recombinant segment was created to minimize the genome size increase in mutants .', 'In this strategy ( Rec II ) , the length of the Ech_0126 segment was reduced to approximately equal the length of the antibiotic resistance cassette introduced .', 'The homologous recombination schemes to achieve this are presented as a cartoon in supplemental Figure 1 ( Figure S1 ) .', 'Independent of the recombination methods used , mutants were identified which persisted up to six days in culture and the presence of antibiotic had no impact on the mutants recovery; the mutants were assessed by Southern blot analysis of the PCR products generated with primers specific to the E . chaffeensis genomic region upstream of the insertion site and the inserted CAT gene sequence ( Figure S2 ) .', 'Insertion junctions were verified by sequencing of the PCR products ( data not shown ) .', 'Similar experiments were performed with gentamicin resistance cassettes and the mutants were detected similarly for six days .', "We reasoned that the lack of persistently growing mutants in culture may have resulted because the genomic region selected for the homologous recombination may be necessary for the bacterium's continued growth .", 'Alternatively , the antibiotic resistance cassettes containing E . coli codon usage may not be optimal for protein expression in E . chaffeensis .', 'We modified the codon usage for the CAT and gentamicin resistance gene cassettes to optimize their expression in E . chaffeensis and the mutational experiments were repeated .', 'The targeted insertion mutants were detected similar to non-codon optimized constructs , but as in previous experiments the mutants survived only for up to eight days ( not shown ) .', 'We expanded the targeted mutational experiments to six genomic sites by utilizing a mobile group II intron-based mutagenesis method ( the TargeTron method ) [25]–[28] .', 'We opted to utilize this method as many studies in recent years reported its application for creating targeted mutations in several Gram-negative and Gram-positive bacteria [29]–[31] .', 'The mobile group II intron was modified for insertions at six genomic locations of E . chaffeensis , including into the coding regions of Ech_0126 , Ech_1136 , and Ech_1143 and into intergenic regions of the genes Ech_0039 and Ech_0040 , Ech_0111 and Ech_0112 , and Ech_0251 and Ech_0252 .', "We inserted the pathogen's tuf promoter to promote expression of modified group II intron RNA and intron encoded protein in E . chaffeensis .", 'The CAT gene cassette was also part of the modified mobile group II introns to confer resistance against chloramphenicol .', 'Multiple assessments of insertions in all of the target regions led to the consistent identification of mutants when the mobile group II intron was targeted to the genes Ech_0126 , Ech_1143 and to the non-coding region between the genes Ech_0111 and Ech_0112 , but not in other three locations .', 'Similar to the observations for the homologous recombination at Ech_0126 gene , the mutants persisted only for up to 8 days in culture .', 'The presence of insertions was confirmed by Southern blot analysis of the PCR products generated with primers specific to insertion segment and to the surrounding genomic regions ( Figure S3 ) .', 'Recent studies have demonstrated that Himar1 transposon mutagenesis can be reliably achieved in a rickettsial organism closely related to E . chaffeensis , i . e . , A . phagocytophilum [32] .', 'In creating random mutations in E . chaffeensis , we utilized Himar1 transposon constructs similar to the ones used for mutagenesis of A . phagocytophilum [32]; the constructs prepared for this study represent single plasmids containing the Himar1 transposase gene and a transposon segment comprising of genes encoding mCherry or green fluorescent protein ( GFPuv ) co-expressed with the aad gene to confer resistance to spectinomycin and streptomycin flanked by inverted repeats recognized by the transposase .', 'Three independent electroporation experiments were performed ( once with the mCherry plasmid and twice with the GFPuv plasmid ) using ISE6 tick cell-derived E . chaffeensis organisms; the organisms were then propagated in both ISE6 cells and DH82 cells .', 'Antibiotic resistant organisms that persisted in culture for several months were identified using dual selection with spectinomycin and streptomycin ( 100 µg/ml each ) .', 'The cultures also expressed mCherry or GFPuv ( Figure 1 ) .', 'The transposon insertions in the mutants were verified by Southern blot analysis performed with an aad gene probe ( Figure 2 ) .', 'Several restriction enzymes lacking recognition sites in the region that bound the DNA probe were used in the analysis .', 'Several insertion sites were observed in the mCherry mutant DNA , as judged from the Southern blot signals ( both weak and strong signals ) .', 'Similarly , multiple insertions were observed in the first GFPuv mutant DNA .', 'In the second transformation experiment with the GFPuv plasmid , we recognized only one strong insertion segment .', 'The intensity and the presence or absence of the hybridized DNA fragments in the mCherry and in the 1st GFPuv plasmid transformants were variable in DNA isolated from the organisms recovered from infected macrophages or tick cells ( Figure 2 , panel A ) .', 'Similarly , hybridized fragments were variable in cultures when harvested at different times following electroporation ( data were presented for the cultures grown in macrophage cell line ) ( Figure 2 , panel B ) .', 'To map the genomic locations of the transposon insertions , inverse PCRs and ST-PCRs ( semi-random , two-step PCR ) followed by sequence analysis of the PCR products were performed .', 'We mapped five genomic locations of the mCherry transposon inserts and four genomic locations from the GFPuv insets ( three from the 1st and one from the 2nd GFPuv transformants ) ( Figure 3 ) .', 'Six of the 9 transposon insertions were present within the non-coding regions of the genome; one of the insertions is close to the termination codon of the gene Ech_0230 ( just 18 base pairs 3′ to the termination codon ) .', 'The remaining three insertions were present within the coding regions of three hypothetical protein encoding genes Ech_0379 , and Ech_0601 , and Ech_0660 .', 'The insertions at all 9 genomic sites were confirmed by PCR and sequence analysis with primers designed to bind to the regions targeting the inserted DNA and to the surrounding genomic locations ( Figure 4 ) .', 'Himar1 transposon insertions occur mostly once or twice per genome [36]–[38] .', 'In this study , we observed multiple insertions in the mCherry transposon mutants and similarly for the first experiment performed with the GFPuv construct .', 'We reasoned that the mutants represented heterogeneous mixture of organisms; this is consistent with varied signals in the Southern blots for the mutants isolated at different times in culture and for the observed differences in mutants recovered from macrophage and tick cell cultures ( Figure 2 ) .', 'To test the hypothesis that the mutants represent a heterogeneous mixture , we performed serial dilution culture experiments with 10 fold dilutions of cell-free organisms mixed into uninfected macrophage cultures , and the infection in higher dilutions ( an estimated number of bacteria in the range of 0 . 1 to 10 per well ) was monitored for growth .', 'We used Southern blotting ( not shown ) and PCR to target all 8 genomic insertions in cultures that tested positive for bacterial growth ( Figure 5 ) .', 'The PCR products were observed for the clonal population only for the insertion downstream to the Ech_0284 ( lane 2c in Figure 5 ) from the mCherry mutant pool following the dilution cloning experiment .', 'Similar efforts in recovering clonal populations from GFPuv mutant cultures resulted in the elimination of one of the three mutants from the pool ( mutant downstream to the Ech_0202 ) ( Lane 6c in Figure 5 ) Three of the 9 transposon mutants included insertions into the protein coding regions of three different hypothetical protein genes; Ech_0379 , Ech_0601 and Ech_0660 .', 'To evaluate if the insertions resulted in the loss of gene activity from these genes , mRNA recovered from the wild-type and mutant organisms grown in culture was assessed by RT-PCR .', 'All three genes are transcriptionally active in wild-type bacteria replicating in macrophages and two of the three genes are also active during growth in AAE2 tick cells ( Ech_0379 and Ech_0601 ) ( Figure 6 ) .', 'Transcript levels for Ech_0379 were , however , low in the tick cell-derived bacteria , whereas Ech_0601 expression levels were similar in both macrophages and tick cells .', 'Transposon insertions into the coding sequences of all three genes resulted in complete loss of gene expression ( Figure 6 ) without impacting growth in macrophage or tick cell cultures in vitro .', 'As the insertion mutation near Ech_0230 is only 18 base pairs downstream from its coding sequence , we also assessed the impact of this mutation on the gene expression .', 'This gene is expressed in wild-type organism grown in both the macrophage and tick cell cultures .', 'The mutation , however , resulted in the loss of gene expression ( Figure 6 ) .', "We investigated whether one or more mutations into the coding or non-coding regions affected the pathogen's growth and persistence in a mammalian host or its acquisition by tick .", 'A laboratory reared , white-tailed deer was inoculated intravenously with a mixed ( i . e . , not a clonal population ) mCherry transformant culture containing all five different insertion mutants .', 'The presence of mutants in the inoculum was confirmed by Southern blot and insertion-specific PCRs ( not shown ) .', 'Deer blood was monitored for infection by culture recovery of the organisms and PCR analysis targeting to all five insertion sites and for the presence of spectinomycin resistance gene for up to 63 days .', 'With the exception of a few post-infection dates , the animal tested positive for the culture recoverable pathogen and insertion-specific , spectinomycin resistance gene ( aad ) ( Table', '1 ) throughout the study period , suggesting persistence of the mutant organisms .', 'The pathogens recovered from deer also expressed functional mCherry protein similar to the mutants in culture prior to infecting the animals ( data not shown ) .', 'Evaluation of deer blood-derived total genomic DNA by insertion-specific PCRs targeting to all five genomic locations originally identified in the mCherry transformants resulted in the amplification of segments only from three intergenic locations ( Table 1 ) .', 'Amplicons diagnostic for the insertion within the coding region of the gene Ech_0379 and one insertion into the non-coding region located 18 base pairs downstream from the Ech_0230 coding sequence were negative in all blood samples analyzed .', 'Furthermore , the culture recovered organisms from blood samples evaluated by insertion-specific PCRs confirmed the presence of mutations only at the same three non-coding regions ( not shown ) .', 'To confirm these results , we repeated the deer-infection experiment using mCherry transformants in two additional animals , and followed the infection for 49 days .', 'As in the first experiment , mutants persisted in deer included only the same three intergenic insertions , whereas transformants with mutations into the coding region of Ech_0379 and the non-coding region mutation at the 3′ end of the Ech_0230 termination codon were not detected ( Table 2 ) .', 'Nymphal A . americanum ticks were allowed to feed on a deer starting on day five post infection; fully engorged ticks that dropped off the animal were allowed to molt .', 'Ten randomly collected molted adult ticks of both sexes were tested for E . chaffeensis transformant DNA by nested PCR targeting the spectinomycin resistance gene .', 'Seven ticks that tested positive for the mutants were further assessed for the presence of mutations at all five genomic insertion sites ( Table 3 ) .', 'The tick DNAs tested PCR positive for insertion mutations in two intergenic locations , and tested negative for the insertion within the coding region of the gene Ech_0379 and the non-coding insertion near Ech_0230 gene coding sequence , as observed from the PCR results for deer blood .', 'The ticks were also negative for one other insertion located in the non-coding region between the genes Ech_0284 and Ech_0285 .', 'The mutant pool infection experiments described above are similar to studies reported in the literature in identifying virulence associated genes by negative selection of several bacterial pathogens [39]–[41]; it will be a valuable tool for identifying additional genes essential for the E . chaffeensis infection in vivo .', 'To further validate that the negative selection approach aids in identifying essential genes of E . chaffeensis , we used two clonal populations of mutants in another infection study in deer .', 'One of these mutants had insertion within the coding region of the gene Ech_0660 and the second one has a mutation in the intergenic region located between the genes Ech_0284 and Ech_0285 .', 'This clonal pair was selected because the Ech_0660 mutant was identified as having single insertion in the genome ( Figure', '2 ) resulting in the loss of gene expression ( Figure 6 ) and the mutant in the intergenic region between Ech_0284 and Ech_0285 is one of the mutants that grew continuously in infected deer when inoculated as part of the pool of mutants ( described above ) .', 'Blood samples collected from the infected animals for up to 20 days were assessed to monitor the mutants in circulation by performing nested PCR analysis ( Table 4 ) .', 'The Ech_0660 infected animals tested negative for blood samples assessed for all post-infection days , whereas the infection with the intergenic mutant clone persisted in deer , as evidenced by the PCR positives on several post-infection days .'], ['Ehrlichia species cause important diseases in people and animals , and understanding the pathogenesis and host-specific differences in gene expression of these pathogens are important goals .', 'However , a major hurdle in advancing research in Ehrlichia species has been the lack of genetic tools , specifically , reliable methods for mutagenesis .', 'This may largely be due to the fact that these organisms can survive only for a very short period of time outside a host cell [20] .', 'Moreover , these phagocytotropic organisms do not appear to harbor plasmids [42] , and may not be amenable to plasmid transformation .', 'Although preliminary evidence resulting from attempts to mutagenize E . chaffeensis have been discussed at scientific meetings [43] , [44] , stable mutants have not been realized until now .', 'In this study , we took a methodical approach in demonstrating the feasibility of creating both targeted and random mutations in E . chaffeensis .', 'Firstly , we evaluated various experimental conditions to identify protocols that work the best in transforming the organism with a plasmid ( results not discussed ) .', 'Secondly , we assessed various antibiotics in support of identifying drugs that are useful in monitoring the mutants .', 'We then tested the feasibility of creating targeted mutations by homologous recombination at a site which appeared to be transcriptionally silent .', 'We subsequently expanded the targeted mutagenesis to six genomic locations using the modified mobile group II intron method , as recent research had demonstrated that it is an efficient method for creating targeted mutations in several Gram positive and Gram negative bacteria [28]–[31] .', 'The method aided in the consistent identification of mutations in three targets assessed , including one located within a differentially expressed gene ( Ech_1143 ) that encodes an outer membrane protein , p28-Omp 19 .', 'The mutants , however , survived in culture only for a short period of up to 8 days .', 'We then evaluated transposon-based random mutagenesis and demonstrated that stable E . chaffeensis mutants could be generated in this way .', 'Random mutagenesis resulted in multiple mutations at several genomic locations .', 'The random mutants included within the coding regions of two differentially expressed genes ( Ech_0379 and Ech_0660 ) encoding for two different hypothetical proteins and one in the coding region of a constitutively expressed hypothetical protein gene ( Ech_0601 ) .', 'Together , the experiments described in this study demonstrate that it is possible to create mutations within E . chaffeensis genes in both constitutively and differentially expressed genes , as well as in intergenic regions .', 'The choice of mutagenesis methods does not seem to have any impact in creating mutations at a specific region of the genome .', 'For example , we used three independent methods in creating targeted insertion mutations into the Ech_0126 gene ( two different approaches of homologous recombination and a TargeTron method ) .', 'The use of all three methods aided in the identification of disruption mutations within this gene .', 'It is not clear why the targeted mutations did not survive longer in culture; several possibilities should be considered:', "1 ) it is possible that the genomic regions selected for insertion mutations may represent regions that are necessary for the organism's normal persistent growth in culture .", '2 ) Alternatively , the E . chaffeensis promoter ( rpsl ) utilized in driving the expression from CAT gene may not be optimal in E . chaffeensis conferring resistance against the antibiotic concentration used .', 'Although the rpsl promoter worked well in developing resistance in E . coli in driving the CAT gene expression ( not shown ) , its expression in E . chaffeensis may be an issue due to duplication of the rpsl promoter sequence in the genome .', 'We are now investigating the use of heterologous promoters in targeted mutation experiments , such as the A . marginale tr promoter , as we already demonstrated its utility in the current study in the transposon mutagenesis in E . chaffeensis .', 'Our efforts to create targeted mutations resulted in the establishment of mutants only in three out of six genomic targets evaluated .', 'We reasoned that the targets selected for the three regions where mutants could not be detected might represent essential regions of the genome .', 'One in particular , the Ech_1136 gene encodes for a protein ( p28-Omp14 ) that is differentially expressed with significant expression being observed in tick cells in vitro and in vivo [15]–[17] , [45] , [46] .', "The p28-Omp 14 is also a highly immunogenic protein; we reasoned that this gene product is an important component and its expression is critical for the organism's survival in a tick cell environment , but may be less critical for its growth in macrophages .", "The mutational analysis , however , suggested that the insertion mutation into this gene results in the complete inhibition of E . chaffeensis growth; this conclusion may infer that this gene product is necessary for the organism's growth in macrophages under in vitro conditions .", 'This hypothesis is consistent with our recent studies suggesting that the protein from this gene is also expressed in macrophages [15] .', 'Interestingly , targeted mutation in the predominantly macrophage-specific outer membrane protein gene of p28-Omp 19 ( Ech_1143 ) , a paralog of the p28-Omp 14 gene , appears to have no impact on the growth of the organism in macrophage cultures .', 'Southern blot analysis of DNA recovered from transposon mutants harvested at different times during their growth in macrophage or tick cell cultures suggested that the mutants are a heterogeneous population .', 'The Himar1 transposase mutations typically occur only at one or two locations in a genome [36]–[38] .', 'To verify that the Himar transposon mutations in E . chaffeensis are similarly occurring at fewer genomic locations , we attempted to clone the mutants by performing a serial dilution cloning experiment .', 'Establishing clonal populations from mutant pools is challenging and is a time consuming task for intracellular pathogens , such as E . chaffeensis .', 'However , we were able to identify a single insertion containing culture from the mCherry mutant population and could eliminate one mutant from the GFPuv mutant pool .', 'We mapped 9 random insertion mutation sites in the E . chaffeensis genome; six were located within non-coding regions and three were present within coding regions of three different transcriptionally active genes , and the mutations within the coding regions inhibited the mRNA production .', 'Two of the active genes disrupted by the Himar1 insertion were differentially expressed genes [expressed highly ( Ech_0379 ) or exclusively ( Ech_0660 ) in macrophage cultures] .', 'The complete inactivation of the genes caused by these mutations did not affect the growth of the organisms in either macrophages or tick cells .', 'The nine genomic locations are the first to be identified as non-essential for E . chaffeensis growth in vitro in both vertebrate and tick cells; however , not all mutants grew in white-tailed deer ( the natural reservoir host for the pathogen [47] ) , and not all were acquired by its transmitting vector tick , A . americanum [47] , [48] .', 'In particular , the transposon insertion into the coding region of the hypothetical protein encoding genes , Ech_0379 and Ech_0660 , and into the non-coding region near the 3′ end of Ech_0230 , prevented growth of the mutant organisms in deer .', 'We were puzzled with the observation that the insertion mutation in a non-coding region also limited the growth of the organism in deer .', 'One possibility is that insertions at certain non-coding regions of a genome may cause a polar effect influencing gene expression from surrounding genes leading to abolished growth in vivo , similar to the observations noted in the literature for other organisms [49] .', 'Our RNA analysis , however , revealed that the mutation at the 3′ end of the ECH_0230 caused the inactivation of gene expression from this gene .', 'The 3′ end mutation may have altered the transcript stability of Ech_0230 , possibly because it may be located within the DNA segment representing the transcriptional unit .', 'We performed the infections in deer with pool of mutants in three separate animals using two independently cultured batches of organisms and the analysis also included 31 blood samples collected at various post-infection days ( 10 or 11 harvest points from each animal ) .', 'All five mutants were represented in the culture pool used for infections .', 'Independent of a blood sample analyzed , the only mutants consistently not detected in deer blood were those with the loss of gene expression ( in genes Ech_0230 and Ech_0379 ) .', 'The in vivo survived mutants in deer , therefore , represent only those for which the transcriptional activity was not negatively affected in genes near the insertion mutations .', 'More importantly , mutations causing the loss of gene expression correlated very well with their failed growth in deer or their non-acquisition by ticks during blood feeding .', 'Together , these data suggest that the elimination of some mutants in vivo from a pool of mutants is the result of the E . chaffeensis growth inhibition because of the loss of gene function probably impacting the survival of the pathogen in vivo .', 'The protocols outlined in this study , beginning from creating mutations and assessing their ability to grow or not to grow in a host ( Figure 7 ) , will serve as a useful tool in screening the genome of E . chaffeensis to identify additional genes contributing to the pathogenesis .', 'The in vivo infection assessment with pools of mutants to identify virulence associated genes in bacterial pathogens is well-documented in the literature [39]–[41] .', 'Our study , however , is the first to apply similar strategy for an obligate intra cellular rickettsial pathogen and it is likely valuable in initiating similar studies in other related pathogens .', 'The method may also be applicable for other obligate intracellular pathogens , such as Chlamydia for which genetic manipulation methods are still at infancy [50] .', 'Establishing clonal populations from mutant pools is a challenging task for intracellular pathogens , such as E . chaffeensis .', 'We , however , were able to prepare two clonal populations from the mutants described in this study; one having mutation causing the loss of gene expression ( Ech_0660 ) and the second one having a mutation into a noncoding region ( between the genes Ech_0284 and Ech_0285 ) with no impact of gene expression in genes 5′ and 3′ to the insertion mutation ( data not shown ) .', 'Infections with the two clones in deer further supported the conclusions drawn from the infection experiments using pool of mutants .', 'The Ech_0660 mutant causing the gene expression inactivation also failed to grow in deer , while the intergenic mutant persisted well .', 'The lack of growth of the subset of mutants from a pool of mutants in deer , therefore , is not a random event because the only mutants that failed to grow in deer are those impacting the gene expression of the organism .', 'Ticks also did not acquire E . chaffeensis mutants containing gene function disruptions .', 'One other mutant with insertion into the non-coding region between the genes Ech_0284 and Ech_0285 also did not grow in ticks .', 'Failure of ticks to acquire the gene disruption mutants is likely due to their inability to productively infect deer during the time of tick feed
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['Ehrlichia chaffeensis is a tick transmitted pathogen responsible for the disease human monocytic ehrlichiosis .', 'Research to elucidate gene function in rickettsial pathogens is limited by the lack of genetic manipulation methods .', "Mutational analysis was performed , targeting to specific and random insertion sites within the bacterium's genome .", 'Targeted mutagenesis at six genomic locations by homologous recombination and mobile group II intron-based methods led to the consistent identification of mutants in two genes and in one intergenic site; the mutants persisted in culture for 8 days .', 'Three independent experiments using Himar1 transposon mutagenesis of E . chaffeensis resulted in the identification of multiple mutants; these mutants grew continuously in macrophage and tick cell lines .', 'Nine mutations were confirmed by sequence analysis .', 'Six insertions were located within non-coding regions and three were present in the coding regions of three transcriptionally active genes .', 'The intragenic mutations prevented transcription of all three genes .', 'Transposon mutants containing a pool of five different insertions were assessed for their ability to infect deer and subsequent acquisition by Amblyomma americanum ticks , the natural reservoir and vector , respectively .', 'Three of the five mutants with insertions into non-coding regions grew well in deer .', 'Transposition into a differentially expressed hypothetical gene , Ech_0379 , and at 18 nucleotides downstream to Ech_0230 gene coding sequence resulted in the inhibition of growth in deer , which is further evidenced by their failed acquisition by ticks .', 'Similarly , a mutation into the coding region of ECH_0660 gene inhibited the in vivo growth in deer .', 'This is the first study evaluating targeted and random mutagenesis in E . chaffeensis , and the first to report the generation of stable mutants in this obligate intracellular bacterium .', "We further demonstrate that in vitro mutagenesis coupled with in vivo infection assessment is a successful strategy in identifying genomic regions required for the pathogen's in vivo growth ."]
['The tick-transmitted bacterium , Ehrlichia chaffeensis , causes human monocytic ehrlichiosis , an acute febrile illness that can progress to a fatal outcome .', 'This and other related pathogens have evolved to establish infections in vertebrate and tick hosts for completing their lifecycle .', "Our recent studies suggest that the pathogen's differential gene expression during growth in ticks and mammals is a major contributor for its dual host adaptation .", 'However , the importance of the pathogen phenotype differences is best understood if we have methods to knock down protein expression from one or more genes .', 'Creating mutations in obligate intracellular pathogens remain a challenge due to their limited survival in the extracellular environment .', 'Here , we present evidence for multiple insertion mutations in the E . chaffeensis genome .', 'Three of the nine mutations in the genome inhibiting gene expression prevented infection of deer , the natural host for the pathogen .', 'This is the first study demonstrating the feasibility of creating mutations in an Ehrlichia species; and directly linking specific regions of the genome to in vivo infection .', 'Methods described here allow for studies to define genes important for infectivity and ability to cause disease , and are equally important for initiating similar studies in other related emerging zoonotic pathogens .']
['microbial mutation', 'vector biology', 'microbial pathogens', 'microbial control', 'genetics and genomics', 'biology', 'genomics', 'microbiology', 'ticks', 'host-pathogen interaction', 'bacterial pathogens', 'microbial growth and development']