Rickettsia typhi Possesses Phospholipase A2 Enzymes that Are Involved in Infection of Host Cells
[['Bacterial species of the genus Rickettsia ( Alphaproteobacteria: Rickettsiales: Rickettsiaceae ) are Gram-negative , obligate intracellular bacteria with a life cycle typically involving arthropod vectors and vertebrate hosts [1] .', 'Some members of the genus Rickettsia are serious human pathogens , such as the agents of epidemic typhus ( R . prowazekii ) and Rocky Mountain Spotted Fever ( R . rickettsii ) [1] .', 'Included with R . prowazekii in the typhus group ( TG ) rickettsiae , R . typhi is the causative agent of murine typhus and is transmitted by fleas throughout the world [2] .', 'Murine typhus presents as a mild to severe flu-like illness , with over 70% of patients requiring hospitalization , and if left untreated , can be fatal in humans [3] , [4] , [5] .', 'Murine typhus is endemic in the continental US and is substantially re-emerging in southern Texas and California , where the current level of reported human cases is continuing to occur with high prevalence [2] , [6] , [7] , [8] , [9] .', 'The obligate intracellular life cycle of Rickettsia spp .', 'involves entry into host cells by phagocytosis ( or induced phagocytosis for non-phagocytic cell types ) , rapid escape from the phagocytic vacuole into the host cytoplasm to evade phagosome-lysosome fusion , replication within the host cytoplasm , and exit from the host cell by actin-mediated motility ( e . g . , Spotted Fever Group rickettsiae ) or lysis of host cells ( e . g . , TG rickettsiae ) [1] , [10] .', 'The genomes of nearly 50 rickettsial species have been sequenced and provide many insights into their biology [11] .', 'However , very little is known regarding the molecular mechanisms of rickettsial intracellular growth and pathogenesis due to limited tools and approaches for genetic manipulation [12] , [13] .', 'Rickettsial phospholipase A2 ( PLA2 ) activity has long been proposed to mediate rickettsial entry into host cells , escape from the phagosome and lysis of host cells [14] , [15] , [16] , [17] , [18] .', 'However , the corresponding rickettsial gene ( s ) encoding PLA2 and the exact mechanism of such enzymes ( e . g . , host/vector range , substrate specificity , and activity period during life cycle ) in rickettsial intracellular life is not well understood .', 'Recently , we reported that the R . typhi genome possesses two genes encoding patatin ( Pat ) -like PLA2 proteins: RT0590 ( Pat1 ) and RT0522 ( Pat2 ) , and demonstrated that Pat2 possesses PLA2 activity [19] .', 'A subsequent report demonstrated that the Pat2 homolog of R . prowazekii ( RP534 ) also possesses PLA2 activity [20] .', 'While Pat1 homologs are encoded in all sequenced Rickettsia genomes , Pat2 is known from a much narrower range of species [19] , [21] .', 'Genes conserved in TG rickettsiae , relative to the larger rickettsial genomes , likely underlay important factors distinguishing TG rickettsiae cell biology and pathogenesis from other rickettsioses [22] .', 'Thus , the potential utilization of two divergent Pat enzymes throughout the bacterial infection cycle may indicate a mechanism unique to TG rickettsiae .', 'Elucidating the role of patatins in rickettsial infection is important for determining the factors underlying rickettsial obligate intracellular infection and pathogenesis .', 'In our previous report [19] we showed that the cytotoxicity and PLA2 activity of R . typhi Pat2 is relatively low compared to that of Pseudomonas aeruginosa ExoU , a secreted cytotoxin and virulence factor with PLA2 activity [23] , [24] .', 'This suggests that R . typhi Pat2 may be necessary to support intracellular survival without affecting host cell integrity , at least until the rickettsiae are ready to lyse the host cell and spread to cause further infection .', 'In this communication , we report the further characterization of PLA2 activity for both Pat1 and Pat2 of R . typhi , and also provide important properties of both enzymes during R . typhi infection of host cells .', 'Using bioinformatics and comparative genomics , important evolutionary characteristics of both patatins are illustrated and suggest differential strategies for patatin utilization across species of Rickettsia .', 'Collectively , these new data shed light on the molecular dynamics orchestrating the R . typhi obligate intracellular life cycle within eukaryotic cells .'], ['Informatics analysis identified two Pat-like phospholipases encoded within Rickettsia genomes , both of which contain the features typical of Pat proteins ( Fig . 1A ) .', 'The rickettsial Pats ( Pat1 and Pat2 ) are larger than the average size of bacterial Pats , with the C-terminal halves of both proteins highly divergent and dissimilar to the C-terminal regions of other prokaryotic and eukaryotic Pats ( data not shown ) .', 'Comparative analysis of 46 Rickettsia genomes indicates that Pat1 is a ubiquitous protein that is likely essential for rickettsial biology ( Fig . 1B ) .', 'Alternatively , Pat2 is encoded in only 24% of Rickettsia genomes , with apparent loss of the pat2 gene occurring multiple times throughout rickettsial evolution .', 'Pseudogenization of pat2 is an active process , as indicated in some genomes by short annotated ORFs with strong homology to portions of full length Pat2 proteins , or by even smaller fragments identified using TBLASTN ( data not shown ) .', 'Rickettsia Pat1 and Pat2 are very divergent from one another .', 'The best scoring matches to the NCBI Conserved Domains Database ( CDD ) for Pat1 and Pat2 are cd07199 and cd07207 , respectively .', 'cd07199 includes PNPLA8 , PNPLA9 , and Pat17 patatin-like phospholipases , while cd07207 is typified by the secreted bacterial proteins ExoU ( Pseudomonas aeruginosa ) and VipD ( Legionella pneumophila ) .', 'Within-genome BLASTP searches ( e . g . , using Pat1 as a query to detect Pat2 , and vice versa ) do not result in significant matches , and inspection of the top 500 hits in BLASTP searches against the NCBI database of non-redundant ( nr ) protein sequences ( using either Pat1 or Pat 2 sequences as queries ) suggest that distant homologs of both proteins do not overlap .', 'This sequence divergence between Pat1 and Pat2 , coupled with their distribution patterns across Rickettsia genomes , suggests different functions for Pat1 and Pat2 in rickettsial biology .', 'The top scoring BLASTP hits against the nr database ( excluding Rickettsia ) for R . typhi Pat1 ( the lycophyte Selaginella moellendorffii , 122 bits , 35% aa identify ) and Pat2 ( L . pneumophila str . Corby , 169 bits , 39% aa identity ) illustrate that both proteins are highly divergent from their closest homologs in other organisms .', 'Phylogeny estimation of Pat1-like sequences placed the rickettsial proteins in a clade with Pats from other intracellular bacterial species , including “Candidatus Odyssella thessalonicensis” ( Rickettsiales ) , Coxiella burnetii ( Gammaproteobacteria ) , “Candidatus Amoebophilus asiaticus” ( Bacteroidetes ) , and Rickettsiales endosymbiont of Trichoplax adhaerens ( Fig . S1 ) .', 'Phylogeny estimation of Pat2-like sequences placed the rickettsial proteins in a clade with ExoU ( Pseudomonas spp . ) , VipD ( Legionella spp . ) and related Pats from other Gammaproteobacteria and Betaproteobacteria species ( Fig . S2 ) .', 'Thus , neither Pat1 nor Pat2 of Rickettsia spp .', 'show a pattern of vertical descent in the bacterial species tree , with Pats and other phospholipases from other Alphaproteobacteria ( including Anaplasmataceae , the sister family to Rickettsia spp . ) being unrelated .', 'The chromosomal locations of both pat1 and pat2 ( full length or pseudogene ) are conserved across all Rickettsia genomes , suggesting both genes were present in the last common ancestor of the analyzed genomes ( data not shown ) .', 'Despite rampant pat2 pseudogenization across many Rickettsia genomes , the 11 full-length Pat2 proteins are conserved in sequence ( 81% avg . aa identity ) with negligible length variation .', 'Phylogeny estimation of the Rickettsia Pat2 sequences is congruous with the Rickettsia species tree ( Fig . S2 ) , suggesting independent gene loss and a lack of recombination at this conserved locus .', 'The absence of pat2 genes in the pool of rickettsial mobile genetic elements ( Rickettsia mobilome ) supports these observations .', 'Alternatively , despite being encoded in all Rickettsia genomes , Pat1 sequences are extraordinarily divergent in sequence length and composition , particularly in the variable ( V ) region that comprises the majority of the C-terminal half of the protein .', 'The largest Pat1 sequences ( R . canadensis genomes ) contain from six to eleven Glu-rich repeats ( EENIENNQETINKPITQALEPDDEDFEE ) within the V region , with smaller cryptic repeats predicted in other Pat1 sequences ( data not shown ) .', 'Phylogeny estimation of Pat1 proteins yielded a tree that is highly incongruent with the Rickettsia species tree ( shown in Fig . 1 ) , a result previously shown in an analysis of only nine Rickettsia genomes [25] .', 'Our robust phylogeny estimation placed the Pat1 sequences from 46 Rickettsia genomes into two well-supported groups ( Pat1A and Pat1B ) ( Fig . 2A ) .', 'The Pat1B sequences include plasmid-encoded Pat1 proteins ( pRF of R . felis and pMCE_1 of “Candidatus R . amblyommii” ) , the larger R . canadensis proteins , and the proteins from REIS , R . helvetica ( incertae sedis ) and the transitional group rickettsiae [26] .', 'The remaining Pat1 proteins form the Pat1A group , which superficially corroborates the Rickettsia species tree ( minus the sequences from Pat1B ) .', 'This phylogeny estimation , coupled with the conserved chromosomal locale of all pat1 genes , strongly implicates multiple recombination events between rickettsial plasmid-encoded pat1 and the chromosomal-encoded pat1 genes within the Pat1B group .', 'Despite being highly divergent from one another , Pat1A and Pat1B proteins contain a conserved C-terminal tail ( T ) containing approximately 100 aa residues ( Fig . 2B ) .', 'This further fortifies the role of recombination in shaping Pat1 diversity across Rickettsia genomes .', 'Six residues within the T region , plus an additional nearby residue in the V region , were predicted to have evolved under positive selection .', 'This implicates a possible function for the T region , perhaps in contact with host cell molecules , or in mediating the secretion of Pat1 out of the rickettsial cell ( see DISCUSSION ) .', 'To determine the expression of Pat1 protein and its secretion into host cells during R . typhi infection , we prepared two cellular fractions , pellet and supernatant , from uninfected or R . typhi-infected Vero76 cells after incubation for 48 h .', 'The pellet contained intact rickettsiae along with host cell debris .', 'The supernatant contained the rickettsial secreted proteins , as well as the host soluble proteins .', 'Both fractions from uninfected or R . typhi-infected Vero76 cells were probed with antibodies raised against Pat1 , Pat2 , rOmpB ( Sca5 ) , EF-Ts or GAPDH .', 'GAPDH , which is a host cytoplasmic protein , was detected in the supernatant of both the uninfected and infected cells ( Fig . 3 , Lanes 2 and 4 ) .', 'The control EF-Ts , a soluble rickettsial cytoplasmic protein , was found only in the pellet of infected cells ( Fig . 3 , Lane 3 ) , supporting that EF-Ts remained associated with R . typhi and that R . typhi remained intact during fractionation .', 'We observed that rOmpB , which is a rickettsial outer membrane protein [27] , was only present in the pellet of infected cells ( Fig . 3 , Lane3 ) .', 'This suggests rOmpB was not stripped and solubilized by 0 . 1% Triton X-100 treatment , but remained associated with the R . typhi cell surface .', 'However , the positive control for R . typhi secreted protein Pat2 [19] and Pat1 were present in both the pellet ( Fig .', '3 , Lane', '3 ) and supernatant ( Fig .', '3 , Lane', '4 ) of infected cells .', 'We also observed that both pellet-associated Pat1 and Pat2 ( containing intact rickettsiae ) migrated slightly slower than supernatant-associated proteins ( containing rickettsial secreted proteins into host cytoplasm ) .', 'These data suggest that Pat proteins are expressed and possibly processed during secretion into the host cell cytoplasm .', 'To further demonstrate the translocation of Pat1 ( as shown in Fig .', '3 ) and Pat2 [19] , we analyzed the subcellular localization of both proteins during R . typhi infection in host cells using immunofluorescence assay ( IFA ) .', 'R . typhi-infected Vero76 cells were immunolabelled with antibodies against Pat1 , Pat2 or R . typhi .', 'While Pat1 and Pat2 co-localized with R . typhi ( Fig . 4A–C and F–H ) , the formation of punctate structures throughout the host cell cytoplasm was also observed , indicating the translocation of both Pat1 and Pat2 from rickettsiae into host cytoplasm .', 'For control , infected cells immunolabelled with preimmune serum ( Fig . 4E ) , as well as uninfected Vero76 cells immunolabelled with anti-Pat antibodies ( Fig . 4D , I ) , did not reveal a similar specific staining .', 'Together , these data suggest that R . typhi Pat1 and Pat2 are translocated from rickettsiae into the host cell cytoplasm during infection .', 'Using a genetically tractable heterologous model system , the yeast Saccharomyces cerevisiae [23] , [28] , [29] , [30] , [31] , [32] , we previously demonstrated the cytotoxicity of R . typhi Pat2 [19] .', 'To determine the potential cytotoxic effect of R . typhi Pat1 , we again implemented the yeast model system .', 'Yeast transformants with Pat1-wt ( carrying the genome sequence of pat1 ) showed unaltered growth on both inducing ( SC-U+Gal ) and repressing ( SC-U+Glu ) agar , as determined by percentage CFU ( Fig . 5A ) .', 'We checked the expression of Pat1-wt in INVSc1 cells under inducing conditions and found no detectable expression of Pat1 protein in yeast cells ( Fig . 5B , Lane 3 ) .', 'As Rickettsia spp .', 'genomes are AT-rich ( the R . typhi genome base composition is 28 . 9% GC ) relative to S . cerevisiae ( 38 . 2% GC ) , we elected to codon optimize pat1 for expression in eukaryotic cells [19] .', 'The yeast strain INVSc1 , transformed with plasmid Pat1-co ( carrying the codon optimized sequence of pat1 ) , expressed the expected protein ( Fig . 5B , Lane 2 ) , showing a substantial reduction in yeast growth ( 25 . 6±5 . 5% ) on inducing agar with respect to repressing agar ( Fig . 5A ) .', 'These data indicate that the expression of Pat1 is cytotoxic to yeast cells .', 'The Ser/Asp catalytic dyad of patatin-like PLA2 enzymes is required for cytotoxicity [19] , [23] , [29] , [30] .', 'We implemented site-directed mutagenesis to examine the contribution of the Ser/Asp catalytic dyad in Pat1-mediated cytotoxicity .', 'The plasmid Pat1-SD was constructed , with Ser-51 ( serine hydrolase motif ) and Asp-199 ( active site aspartate ) residues of pat1-co both replaced with alanine .', 'The yeast strain INVSc1 , transformed with plasmid Pat1-SD , expressed the expected proteins ( Fig . 5B , Lane 1 ) , with S . cerevisiae growth nearly completely restored ( 97 . 7±8 . 1% ) as determined by the CFU assay ( Fig . 5A ) .', 'Collectively , these data indicate that the cytotoxicity caused by the expression of Pat1 in yeast is abolished by mutation at the predicted Ser/Asp catalytic sites of Pat1 , supporting the inference that R . typhi Pat1 is a functional patatin .', 'To determine phospholipase A ( PLA ) activity of Pat1 , we produced recombinant protein Pat1 in an E . coli protein expression system .', 'We also used recombinant Pat2 as a positive control [19] .', 'We observed that the recombinant protein ( Pat1 or Pat2 ) exhibited elevated PLA activity in the presence of Vero76 cell lysate ( Fig . 6A ) , indicating that Pat1 requires a eukaryotic activator for PLA activity .', 'This is consistent with our previous results for Pat2 [19] , although the PLA activity of Pat1 is slightly less than that of Pat2 .', 'We further aimed to identify a possible eukaryotic factor required for the activation of R . typhi Pat1 and Pat2 .', 'The bovine liver superoxide dismutase ( SOD ) has previously been reported to function as an activator of P . aeruginosa ExoU [33] .', 'Therefore , we evaluated the impact of SOD on Pat1 and Pat2 function .', 'The results suggest that bovine liver SOD activates Pat1 and Pat2 PLA activity ( Fig . 6B ) , with the level of activation similar to that observed by Vero76 cell lysate ( Fig . 6A ) .', "Furthermore , the pattern of activation by SOD for both enzymes' PLA activity ( Pat2>Pat1 ) is consistent with that observed by host cell lysate .", 'To examine the effect of the Ser/Asp catalytic residues in Pat1 mediated PLA activity , we produced the mutant derivative protein Pat1-SD ( described above for the yeast model ) in an E . coli protein expression system .', 'The Pat1-SD recombinant protein exhibited significant ( P<0 . 05 [two-tail t-test] ) decrease of PLA activity ( Fig . 6A ) .', 'These data , which showed the effect of mutagenesis of catalytic Ser/Asp sites on PLA activity , clearly suggest Pat1 is a PLA enzyme .', 'To determine whether the inhibitors of calcium-independent ( iPLA2 ) and calcium-dependent cytosolic ( cPLA2 ) PLA2 activity block Pat1 function , we evaluated Pat1 PLA activity in the presence of methyl arachidonyl fluorophosphonate ( MAPF ) , an irreversible inhibitor of both iPLA2 and cPLA2 [19] , [30] , [34] , .', 'At a concentration of 0 . 5 µM , MAPF significantly ( P<0 . 05 [two-tail t-test] ) reduced the PLA activity of Pat1 ( Fig . 6A ) .', 'These data further indicate that Pat1 possesses PLA activity , as this function can be blocked by iPLA2 and cPLA2 inhibitors .', 'We sought to determine the role of Pat1 and Pat2 in R . typhi infection of Vero76 cells by neutralizing their function using anti-Pat1 and anti-Pat2 antibodies .', 'The viability of anti-Pat antibody-treated rickettsiae was quantitatively determined using a plaque assay .', 'This analysis clearly demonstrated that the antibody-treated R . typhi decreased rickettsial infectivity and survival as compared to that by pre-immune IgG treated R . typhi ( Fig . 7A ) .', 'Plaque formation was significantly reduced in anti-Pat1 and anti-Pat2 pretreated groups as compared to the preimmune IgG treated group ( Fig . 7A ) and showed no apparent difference in plaque size for antibody treated rickettsiae with respect to preimmune IgG treated rickettsiae .', 'Similarly , in a separate experiment , we removed excess antibodies and unattached rickettsiae from the host cells by washing prior to the plaque assay .', 'These data showed that R . typhi plaques were decreased to 29 . 27%±2 . 33% or 35 . 49%±3 . 3% for anti-Pat2 or anti-Pat1 antibody , respectively , as compared to that by pre-immune IgG ( Fig . 7B ) .', 'The data also indicated a similar decrease in R . typhi plaque formation whether or not the excess antibody and unattached rickettsiae were removed before addition of agar for the plaque assay ( Fig . 7A , B ) .', 'Together , these data indicate that R . typhi Pat1 and Pat2 are involved at the early stage of R . typhi infection of host cells .', 'Furthermore , an indirect immunofluorescent antibody assay was used to demonstrate the involvement of Pat1 and Pat2 in R . typhi infection of host cells via counting the percent of host cells infected by R . typhi pretreated with anti-Pat1 or anti-Pat2 with respect to those treated with preimmune IgG ( See Materials and Methods ) .', 'These data showed the significant decrease in infection of Vero76 cells by R . typhi pretreated with anti-Pat1 or anti-Pat2 antibodies as determined by IFA ( Fig . 8 ) .', 'Thus , the IFA results suggest that pretreatment of R . typhi with anti-Pat2 or anti-Pat1 antibody blocks rickettsial invasion of host cells , confirming the role of Pat1 and Pat2 during the early stage of R . typhi infection of host cells .', 'Long hypothesized to be mediated by PLA activity [16] , rickettsial phagosome escape was previously shown to occur between 30 to 50 min postinfection [36] .', 'Accordingly , we examined the role of Pat1 and Pat2 in R . typhi phagosome escape into the host cytoplasm during the early stage of infection .', 'Vero76 cells were infected with R . typhi pretreated with anti-Pat1 , anti-Pat2 or preimmune IgG .', 'At 30 min postinfection , rickettsial phagosome escape was monitored by IFA using the endosomal/lysosomal marker lysosomal-associated membrane protein 1 ( LAMP-1 ) .', 'We observed that R . typhi pretreated with anti-Pat1 ( Fig . 9A , panels a to c ) or anti-Pat2 ( Fig . 9A , panels d to f ) antibodies mostly colocalized with the LAMP-1 marker , suggesting the rickettsiae are still enclosed in the phagosome at 30 min postinfection .', 'However , R . typhi pretreated with preimmune IgG are relatively less LAMP-1 positive at 30 min postinfection ( Fig . 9A , panels g to i ) , supporting rickettsial escape from phagosome .', 'Further quantification revealed that the percentage of R . typhi in LAMP-1 positive phagosome was significantly higher for rickettsiae pretreated with anti-Pat1 or anti-Pat2 antibody compared to that with pre-immune IgG ( Fig . 9B ) .', 'Together , these data suggest that antibody neutralization of Pat1 or Pat2 prior to host cell infection blocks or delays R . typhi phagosome escape .'], ['In an earlier report , we showed that pat2 ( RT0522 ) , but not pat1 ( RT0590 ) , is transcribed at all stages of R . typhi intracellular growth in Vero76 cells [19] .', 'This led to our characterization of Pat2 as a functional patatin that is secreted into the host cytoplasm during rickettsial infection .', 'However , the presence of pat1 in all sequenced Rickettsia genomes , coupled with the pseudogenization of pat2 in 76% of these genomes , prompted us to delve deeper at a possible role for the ubiquitous Pat1 in rickettsial biology .', 'In this study , we showed the expression of Pat1 protein in R . typhi cultured in Vero76 cells , despite the inability to detect pat1 mRNA [19] under the same experimental conditions .', 'This phenomenon is perhaps best explained by a potential difference in lifespan for pat1 mRNA and protein .', 'For example , some studies have shown that a lack of correlation between mRNA and protein abundance can be attributed to longer half-lives of protein ( longer than cell cycle ) relative to mRNA ( a few minutes ) in some prokaryotes [37] , [38] .', 'Importantly , labile transcripts relative to protein lifespan may be particularly common for genes activated infrequently relative to workhorse genes ( e . g . , replication and translational machinery genes , basic metabolic genes , etc . ) , resulting in gross underestimation of gene/protein function in studies based solely on transcript ( and not protein ) detection .', 'The present results supporting the function of Pat1 in rickettsial biology are corroborated by the ubiquity of the pat1 gene in 46 Rickettsia genomes , which include species and strains ranging from pathogenic to non-pathogenic ( Fig . 1B ) .', 'The presence in the rickettsial mobilome of plasmid-encoded pat1 genes , and their likely recombination with chromosomal pat1 genes multiple times throughout Rickettsia evolution , strongly imply an essential function for Pat1 in the rickettsial intracellular life cycle .', 'Phylogeny estimation grouped rickettsial Pat1 proteins in a clade with other bacteria possessing an intracellular lifestyle , including Coxiella burnetii , “Candidatus Odyssella thessalonicensis” , “Candidatus Amoebophilus asiaticus” , and Rickettsiales endosymbiont of Trichoplax adhaerens ( Fig . S1 ) .', 'The latter three genomes encode multiple Pat1 homologs , with an extraordinary array of patatins encoded in the “Candidatus Amoebophilus asiaticus” genome [39] .', 'Other closely related clades also contained bacteria with an intracellular lifestyle , especially members of Bacteroidetes .', 'This implies that PNPLA8 , PNPLA9 , and Pat17 patatin-like phospholipases may be common proteins shared by intracellular bacteria , orchestrating functions such as the internalization process , phagosomal escape , and host cell lysis for cell-to-cell spread .', 'Importantly , our phylogeny estimations for both Pat1 and Pat2 proteins suggests a substantial degree of lateral gene transfer ( LGT ) responsible for the origin of Pat genes in Rickettsia spp .', 'and other bacteria , a result consistent with the origin of PLA2s in P . aeruginosa ( ExoU ) [40] and Group A Streptococcus ( SlaA ) [41] via LGT .', 'Our prior study determined that R . typhi Pat2 is secreted into the host cytoplasm during infection , a result consistent with a bioinformatics-based prediction of Pat2 as a secreted protein [19] .', 'However , Pat2 is not predicted to encode an N-terminal signal peptide sequence , suggesting the involvement of a secretion pathway outside of Sec-dependent translocation .', 'Similarly to Pat2 , our bioinformatics analysis [42] , [43] , [44] predicts Pat1 to be noncytoplasmic with no Sec-dependent signal peptide sequence .', 'In this report , by IFA , we have shown that both Pat1 and Pat2 are co-localized with R . typhi , and also form punctate structures throughout the host cell cytoplasm , indicating the translocation of Pat1 and Pat2 from rickettsiae into the host cytoplasm .', 'Data from immunoblotting analysis also revealed the translocation of Pat2 ( positive control ) and Pat1 into the host cytoplasm .', 'However , the slightly slower mobility of rickettsiae-associated Pat1 and Pat2 proteins versus Pat1 and Pat2 proteins that are translocated into the host cytoplasm might be due to the cleavage of a short signal sequence during translocation .', 'While currently unknown , a potential processing mechanism ( cleavage or modification ) for Pat1 and Pat2 during or after translocation from the rickettsial cell will likely emerge via the identification of the apposite secretion pathway ( s ) for these proteins .', 'Insight into the secretion pathway responsible for Pat1 and Pat2 delivery to host cells comes from consideration of other secreted bacterial Pats .', 'The highly cytotoxic ExoU ( P . aeruginosa ) is secreted via the type III secretion system ( T3SS ) [23] , [45] , while the VipD Pat of Legionella spp .', 'is secreted via the I-like ( Dot/Icm ) T4SS [46] .', 'Rickettsia spp .', 'do not encode a T3SS , but do encode a P-like T4SS termed the Rickettsiales vir homolog ( rvh ) T4SS [47] , [48] .', 'Substrates translocated by both I- and P-like T4SSs often contain C-terminal signals that mediate their translocation out of the bacterial cytoplasm and into the extracellular milieu ( or host cytoplasm ) [49] , [50] , [51] , [52] .', 'The conserved nature of the C-terminal sequences of both Pat1 and Pat2 , coupled with our detection of sites evolving under positive selection within the T region of Pat1 ( Fig . 2B ) , suggest these proteins may be rvh T4SS substrates .', 'Alternatively , T1SS substrates also possess C-terminal secretion signals [53] , and Rickettsia spp .', 'encode the OM pore component of T1SSs ( TolC ) and a series of membrane fusion and ABC transporter protein pairs that probably all assemble as T1SSs [26] .', 'While the T1SS in Rickettsia spp .', 'has not been demonstrated to secrete protein , protein substrates from the closely related Ehrlichia chaffeensis ( Rickettsiales: Anaplasmataceae ) have recently been demonstrated to be secreted via the T1SS [54] .', 'Additionally , our very recent work suggests that an ankyrin domain-containing protein of Rickettsia spp .', 'is exported extracellularly via a non-canonical secretion pathway that utilizes both the Sec translocon and TolC , suggesting that TolC has a function in Rickettsia protein secretion [55] .', 'Accordingly , in the future we will investigate both the rvh T4SS and the T1SS for putative roles in Pat1 and Pat2 secretion .', 'Both ExoU of P . aeruginosa [23] , [24] and Pat2 of R . typhi [19] have been reported to be cytotoxic to mammalian and yeast cells , and their PLA activity is required for cytotoxicity .', 'Our data show that the expression of R . typhi Pat1 is also cytotoxic in yeast cells , requiring Ser/Asp catalytic sites for PLA activity and cytotoxicity .', 'Our current data on Pat1 and previously reported data on Pat2 [19] show that the cytotoxicity and PLA activity of these two R . typhi enzymes are relatively low compared to that of ExoU .', 'These data support our earlier interpretation [19] that the low PLA activity is required to support obligate intracellular growth of R . typhi in host cells without inflicting any rapid damage ( at least until bacteria lyse host cells to promote cell to cell spread ) .', 'Importantly , the minimal ( pat2 ) and deficient ( pat1 ) detection of R . typhi Pat transcripts correlates with a milder cytotoxic phenotype of Pat1 and Pat2 relative to the highly potent ExoU .', 'The relatively higher cytotoxic effect and PLA activity of Pat2 [19] compared to Pat1 reported here suggest that Pat1 and Pat2 might be interacting with host cells differently .', 'As discussed above , Pat1 and related PNPLA8 , PNPLA9 , and Pat17 patatin-like phospholipases , are encoded by many intracellular bacteria , not all of which are pathogens .', 'These enzymes may have less of a cytotoxic effect on host cells if they are associated with functions that benefit the bacteria yet do not substantially impact the fitness of the host .', 'Alternatively , the closest homologs of Pat2 that have been functionally characterized , namely ExoU ( Pseudomonas spp . ) and VipD ( Legionella spp . ) , are bona fide virulence factors .', 'The potency of ExoU is demonstrated by its destruction of cellular membranes of mammalian cells , which causes rapid necrotic death [23] , as well as its role in the killing of environmental amoeba [56] .', 'Induced during stationary phase , the most virulent stage of the L . pneumophila life cycle , VipD interferes with vesicular trafficking and promotes intracellular growth [46] , [57] .', 'Thus , it is reasonable to suggest that Pat2 may have a function that is more detrimental to host cells than Pat1 , possibly in the lysis of host cells for cell-to-cell spread .', 'Dependence on eukaryotic activator ( s ) for PLA2 activity has been reported for P . aeruginosa ExoU [23] , [30] , and it has been shown that certain preparations of Cu2+ , Zn2+-SOD1 act as a cofactor to activate ExoU phospholipase activity [33] .', 'A recent report on the Pat2 homolog RP534 of R . prowazekii demonstrated the stimulation of RP534 PLA activity by SOD1 [20] .', 'Our current data on Pat1 and our previous report on Pat2 [19] demonstrate their PLA activities in the presence of Vero76 cell lysate .', 'Our further analysis of R . typhi Pat1 and Pat2 proteins reveal that bovine liver SOD1 activates their PLA activities .', 'The level of activation by bovine liver SOD1 is similar to that by Vero76 cell lysate , indicating the similarity in the enzymatic activation for bacterial patatin-like phospholipases .', 'A recent report by Anderson et al [58] on enzymatic activation of ExoU showed that several ubiquitin isoforms , as well as a subpopulation of ubiquitylated SOD1 and ubiquitylated mammalian proteins , activate ExoU .', 'Although , we have shown here that R . typhi Pat1 and Pat2 proteins required eukaryotic activator ( s ) ( Vero76 cell lysate or bovine liver SOD1 ) for their PLA activities , further analysis of enzymatic activator for Pat1 and Pat2 will be addressed in our future work .', 'The role of PLA2 has long been hypothesized to be involved in rickettsial infection of host cells and phagosome escape [15] , [16]; however , this enzymatic activity has never been demonstrated within the known rickettsial proteome .', 'In this report , we have shown by IFA that pretreatment of R . typhi with anti-Pat1 or anti-Pat2 antibodies negatively affects rickettsial phagosome escape .', 'Furthermore , we have demonstrated by plaque assay and IFA that pretreatment of R . typhi with anti-Pat1 or anti-Pat2 antibodies significantly decreases R . typhi infectivity into Vero76 cells .', 'However , the effect of anti-Pat1 or anti-Pat2 pretreatment on R . typhi infectivity into Vero76 cells is relatively low as determined by IFA compare to that by plaque assay .', 'This disparity in R . typhi infectivity might have resulted due to the difference in our assays for R . typhi infectivity into Vero76 cells .', 'By IFA , we measured the total R . typhi infected host cells , resulting in higher infectivity compared to 
['Introduction', 'Results', 'Discussion', 'Materials and Methods']
['The long-standing proposal that phospholipase A2 ( PLA2 ) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase ( Pat2 ) with PLA2 activity from the pathogens Rickettsia prowazekii and R . typhi .', 'However , pat2 is not encoded in all Rickettsia genomes; yet another uncharacterized patatin ( Pat1 ) is indeed ubiquitous .', 'Here , evolutionary analysis of both patatins across 46 Rickettsia genomes revealed', '1 ) pat1 and pat2 loci are syntenic across all genomes ,', '2 ) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences ,', '3 ) pat2 has been pseudogenized multiple times in rickettsial evolution , and', '4 ) ubiquitous pat1 forms two divergent groups ( pat1A and pat1B ) with strong evidence for recombination between pat1B and plasmid-encoded homologs .', 'In light of these findings , we extended the characterization of R . typhi Pat1 and Pat2 proteins and determined their role in the infection process .', 'As previously demonstrated for Pat2 , we determined that', '1 ) Pat1 is expressed and secreted into the host cytoplasm during R . typhi infection ,', '2 ) expression of recombinant Pat1 is cytotoxic to yeast cells ,', '3 ) recombinant Pat1 possesses PLA2 activity that requires a host cofactor , and', '4 ) both Pat1 cytotoxicity and PLA2 activity were reduced by PLA2 inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues .', 'To ascertain the role of Pat1 and Pat2 in R . typhi infection , antibodies to both proteins were used to pretreat rickettsiae .', 'Subsequent invasion and plaque assays both indicated a significant decrease in R . typhi infection compared to that by pre-immune IgG .', 'Furthermore , antibody-pretreatment of R . typhi blocked/delayed phagosomal escapes .', 'Together , these data suggest both enzymes are involved early in the infection process .', 'Collectively , our study suggests that R . typhi utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle , a mechanism distinguishing it from other rickettsial species .']
['Typhus Group ( TG ) rickettsiae include Rickettsia typhi and R . prowazekii , the etiological agents of murine and epidemic typhus , respectively .', 'The rickettsial obligate intracellular life cycle complicates conventional approaches to genetic manipulation , impeding characterization of molecules underlying pathogenesis .', 'Phospholipase A2 ( PLA2 ) enzymes are factors that likely mediate such processes as host entry , phagosomal escape , and host lysis for cell-to-cell spread .', 'Here , we demonstrate across 46 Rickettsia genomes that genes encoding two PLA2 enzymes ( pat1 and pat2 ) have divergent evolutionary histories and discordant selective constraints , with pat2 deleted from most other non-TG rickettsiae genomes .', 'As previously determined for Pat2 , we show here that R . typhi Pat1 is secreted into the host cell cytoplasm during infection and requires a host cofactor for enzymatic activity .', 'Like Pat2 , recombinant Pat1 protein is cytotoxic to yeast cells .', 'Pretreatment of R . typhi with anti-Pat1 or anti-Pat2 antibody results in a significant decrease in rickettsial infection , implicating roles for both Pat1 and Pat2 during the early stage of R . typhi host cell infection .', 'Collectively , our work suggests that R . typhi utilizes two evolutionary divergent phospholipases during its intracellular life cycle , a mechanism distinguishing TG rickettsiae-associated cell biology and pathogenesis from other rickettsioses , particularly those associated with Spotted Fever Group pathogens .']
['evolutionary biology', 'molecular cell biology', 'biology', 'microbiology']