Comparison of the A2 Gene Locus in Leishmania donovani and Leishmania major and Its Control over Cutaneous Infection

In Old World Leishmania infections, Leishmania donovani is responsible for fatal visceral leishmaniasis, and L. major is responsible for non-fatal cutaneous leishmaniasis in humans. The genetic differences between these species which govern the pathology or site of infection are not known. We have therefore carried out detailed analysis of the A2 loci in L. major and L. donovani because A2 is expressed in L. donovani but not L. major, and A2 is required for survival in visceral organs by L. donovani. We demonstrate that although L. major contains A2 gene regulatory sequences, the multiple repeats that exist in L. donovani A2 protein coding regions are absent in L. major, and the remaining corresponding A2 sequences appear to represent non-expressed pseudogenes. It was possible to restore amastigote-specific A2 expression to L. major, confirming that A2 regulatory sequences remain functional in L. major. Although L. major is a cutaneous parasite in rodents and humans, restoring A2 expression to L. major inhibited its ability to establish a cutaneous infection in susceptible BALB/c or resistant C57BL6 mice, a phenotype typical of L. donovani. There was no detectable cellular immune response against L. major after cutaneous infection with A2-expressing L. major, suggesting that the lack of growth was not attributable to acquired host resistance but to an A2-mediated suppression of parasite survival in skin macrophages. These observations argue that the lack of A2 expression in L. major contributed to its divergence from L. donovani with respect to the pathology of infection. In Old World Leishmania infections, Leishmania donovani is responsible for fatal visceral leishmaniasis, and L. major is responsible for non-fatal cutaneous leishmaniasis in humans. The genetic differences between these species which govern the pathology or site of infection are not known. We have therefore carried out detailed analysis of the A2 loci in L. major and L. donovani because A2 is expressed in L. donovani but not L. major, and A2 is required for survival in visceral organs by L. donovani. We demonstrate that although L. major contains A2 gene regulatory sequences, the multiple repeats that exist in L. donovani A2 protein coding regions are absent in L. major, and the remaining corresponding A2 sequences appear to represent non-expressed pseudogenes. It was possible to restore amastigote-specific A2 expression to L. major, confirming that A2 regulatory sequences remain functional in L. major. Although L. major is a cutaneous parasite in rodents and humans, restoring A2 expression to L. major inhibited its ability to establish a cutaneous infection in susceptible BALB/c or resistant C57BL6 mice, a phenotype typical of L. donovani. There was no detectable cellular immune response against L. major after cutaneous infection with A2-expressing L. major, suggesting that the lack of growth was not attributable to acquired host resistance but to an A2-mediated suppression of parasite survival in skin macrophages. These observations argue that the lack of A2 expression in L. major contributed to its divergence from L. donovani with respect to the pathology of infection. Leishmania protozoa are the causative agents of human leishmaniasis, which is a serious infectious disease throughout the developing world (1Herwaldt B. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar). Leishmaniasis includes a wide range of pathologies ranging from severe lethal visceral infections to self-healing cutaneous lesions, and the specific pathologies are closely related to the species of Leishmania (2Berman J. Clin. Infect. Dis. 1997; 24: 684-703Crossref PubMed Scopus (715) Google Scholar). For example, L. donovani causes visceral leishmaniasis, known as Kala Azar in India and the Sudan, which is fatal if not successfully treated and is characterized by irregular bouts of fever, substantial weight loss, swelling of the spleen and liver, and anemia (1Herwaldt B. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 2Berman J. Clin. Infect. Dis. 1997; 24: 684-703Crossref PubMed Scopus (715) Google Scholar). In comparison, L. major infections, common throughout the Middle East, result in cutaneous lesions that usually heal spontaneously (1Herwaldt B. Lancet. 1999; 354: 1191-1199Abstract Full Text Full Text PDF PubMed Scopus (1403) Google Scholar, 2Berman J. Clin. Infect. Dis. 1997; 24: 684-703Crossref PubMed Scopus (715) Google Scholar). The molecular differences between the different species of Leishmania that govern the outcome of infection and pathogenesis in the human host are unknown. Leishmania protozoa are transmitted to humans through the bite of an infected sandfly. In the sandfly vector, Leishmania exists as flagellated promastigotes, and once they enter the mammalian host after a blood meal, they differentiate into the non-flagellated amastigotes, where they multiply exclusively in the phagolysosomal compartment of macrophages (3Matlashewski G. Farrell J. Leishmania, World Class Parasites series. 4. Kluwer Academic Press, Dordrecht, the Netherlands2002: 105-113Google Scholar, 4Alexander J. Russell D.G. Adv. Parasitol. 1992; 31: 175-254Crossref PubMed Scopus (278) Google Scholar). Differentiation from promastigotes to amastigotes is a critical step for the establishment of infection in macrophages, and therefore genes that are specifically expressed in the amastigote stage are likely to be essential for survival in the mammalian host. The A2 gene family was first identified in L. donovani by virtue of its expression, which is specific to the amastigote stage of the life cycle (5Charest H. Matlashewski G. Mol. Cell. Biol. 1994; 14: 2975-2984Crossref PubMed Scopus (140) Google Scholar), and it has been established that the A2 protein is among the few widely accepted amastigote-specific molecular markers identified to date (reviewed in Ref. 6Gupta N Goyal N. Rastogi A. Trends Parasitol. 2001; 17: 150-153Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The A2 protein represents a virulence factor because L. donovani amastigotes deficient in A2 protein through the use of antisense RNA (7Zhang W.W. Matlashewski G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8807-8811Crossref PubMed Scopus (134) Google Scholar) or gene knock-outs (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar) are attenuated with respect to survival in visceral organs of infected mice. Moreover, intravenous injection of L. major genetically engineered to express A2 produced higher infection levels in the spleen than control L. major, further supporting the argument that A2 enhances survival in resident macrophages of visceral organs (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar). Multiple copies of the A2 gene are clustered on the 850-kb chromosome of L. donovani, which encode a family of A2 proteins, each containing a secretory leader sequence followed by a 10-amino acid sequence that is repeated from 40 to >90 times, depending on the individual A2 gene (5Charest H. Matlashewski G. Mol. Cell. Biol. 1994; 14: 2975-2984Crossref PubMed Scopus (140) Google Scholar, 9Charest H. Zhang W.W. Matlashewski G. J. Biol. Chem. 1996; 271: 17081-17090Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 10Zhang W.W. Charest H. Ghedin E. Matlashewski G. Mol. Biochem. Parasitol. 1996; 78: 79-90Crossref PubMed Scopus (122) Google Scholar). Consistent with the A2 gene family structure and expression, a family of A2 proteins ranging from 42 to 100 kDa is specifically expressed in amastigotes but not in promastigotes (7Zhang W.W. Matlashewski G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8807-8811Crossref PubMed Scopus (134) Google Scholar, 10Zhang W.W. Charest H. Ghedin E. Matlashewski G. Mol. Biochem. Parasitol. 1996; 78: 79-90Crossref PubMed Scopus (122) Google Scholar). Anti-A2 antibodies have been detected in >90% of the sera samples from visceral leishmaniasis patients, confirming that the A2 proteins are expressed in the human host (11Ghedin E. Zhang W.W. Charest H. Sundar S. Kenney R.T. Matlashewski G. Clin. Diagn. Lab. Immunol. 1997; 4: 530-535Crossref PubMed Google Scholar). Preliminary karyotype analysis by Southern blotting revealed that L. donovani A2 protein coding sequences are not present in the genome of all Leishmania species and, notably, appeared to be absent in L. major and L. tropica (11Ghedin E. Zhang W.W. Charest H. Sundar S. Kenney R.T. Matlashewski G. Clin. Diagn. Lab. Immunol. 1997; 4: 530-535Crossref PubMed Google Scholar). Few Leishmania species-specific genes have been identified, and it was therefore possible in the present study to define the genetic basis for the difference in A2 gene structure between L. donovani and L. major. Interestingly, L. major does contain A2 genes, but they are not expressed and lack the multiple repeats in the protein coding regions and therefore represent nonexpressed pseudogenes. Because L. major causes cutaneous infections, it was therefore possible to examine the phenotype associated with restored A2 expression on cutaneous infection and the host cellular immune response to A2-expressing L. major. The results of this study suggest that the inability of L. major to express A2 has contributed to its tropism for cutaneous infections. Mice—BALB/c mice and C57BL/6 (B/6) from or from the of mice in the Leishmania and cutaneous infection in BALB/c mice and L. major and L. donovani L. major and L. donovani promastigotes in with The L. donovani as amastigotes in J. Parasitol. PubMed Scopus Google Scholar). L. donovani into amastigotes was by the promastigotes to amastigote in which the host compartment as J. Parasitol. PubMed Scopus Google Scholar). L. major in the amastigote and this in which to and in this study have been (7Zhang W.W. Matlashewski G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8807-8811Crossref PubMed Scopus (134) Google Scholar, 10Zhang W.W. Charest H. Ghedin E. Matlashewski G. Mol. Biochem. Parasitol. 1996; 78: 79-90Crossref PubMed Scopus (122) Google Scholar). The for was as W.W. Charest H. Matlashewski G. PubMed Scopus Google Scholar). The L. major promastigotes and in containing of The for all and mice infection and and analysis as (7Zhang W.W. Matlashewski G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8807-8811Crossref PubMed Scopus (134) Google Scholar). The as and from L. donovani A2 H. Zhang W.W. Matlashewski G. J. Biol. Chem. 1996; 271: 17081-17090Abstract Full Text Full Text PDF PubMed Scopus (122) Google and from E. Charest H. Matlashewski G. Mol. Biochem. Parasitol. PubMed Scopus Google the from L. donovani gene (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google and the and from L. donovani gene (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar). For A2 protein the was as (7Zhang W.W. Matlashewski G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8807-8811Crossref PubMed Scopus (134) Google Scholar). in cutaneous BALB/c mice infected with promastigotes in was by of infected and The amastigotes in and the Leishmania parasite by promastigotes to amastigote and to For cutaneous infection in C57BL/6 mice and L. major promastigotes, L. donovani and control L. major in with 100 of 100 of 40 of and of promastigotes from of by in Parasitol. 2001; PubMed Scopus Google Scholar). in Parasites into the a in a of The of the was by the of the of the with a in the as S. G. J. N. E. J. J. PubMed Scopus Google Scholar). the and of the infected in containing 100 of 100 of and into and a in a containing 100 of The a and in a containing of containing of blood and with of The of in each was from the which promastigotes be out after of The of was in the The and a and as For in from in containing and was in with or L. and in by to the and the and macrophages in the of of factor or with for and a of promastigotes from the different and infected a of from the a and with in and for with of the with and with to the of infection. In the of infected was and it was among the a of infected of mice with of or in the in a of and and from mice and with and in with and to out of the for The remaining was with in and through a in and from to and the out of the and in For the analysis of the of and and a for the of on the The of that spontaneously out of the was as the of in the by the of in compartment and of the A2 in L. donovani and L. genetic differences have been between L. donovani and L. major, and therefore a of the A2 gene loci an to a detailed of a gene family that between these Old World Leishmania Because genes the A2 genes and are between L. donovani and L. major (11Ghedin E. Zhang W.W. Charest H. Sundar S. Kenney R.T. Matlashewski G. Clin. Diagn. Lab. Immunol. 1997; 4: 530-535Crossref PubMed Google Scholar), an L. donovani was to L. major and from chromosome and is in was for in for comparison, the A2 gene family in L. donovani are clustered in with the gene family and is than chromosome as for L. donovani (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar). in with L. major, the sequence revealed that the of the A2 and genes between L. donovani and L. major. It of that L. major contain A2 gene sequences and that the regions as and of the gene between L. donovani and L. major Moreover, as with L. donovani, is than of A2 sequence chromosome in L. major. are copies of the A2 gene sequences in L. donovani than in L. major. The difference was that the A2 protein coding sequences in the absent in the A2 genes in L. major. a the 10-amino acid of the that are present in all of the L. donovani A2 genes absent in L. major. 10-amino acid in the L. major A2 genes by the leader sequence and a the corresponding L. major A2 protein be of which the first are a functional leader although of the A2 protein coding sequence was absent in the A2 genes in L. major, the and the of the RNA >90% between L. donovani and L. major. is because protein coding sequences remain than the sequences, but the is L. major and L. donovani A2 Southern and analysis of L. major and the A2 from L. major that L. major has the of the A2 genes in and and does not have the multiple copies of the genes as in L. donovani not these revealed and differences in the gene in L. major and L. donovani, where L. major has copies of the A2 genes, and the of the protein coding sequences the remaining A2 genes are of A2 in L. donovani and L. analysis was to the expression of the A2 gene family in L. donovani and L. major specific to the (5Charest H. Matlashewski G. Mol. Cell. Biol. 1994; 14: 2975-2984Crossref PubMed Scopus (140) Google Scholar, W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar), the A2 genes expressed in L. donovani amastigotes and are expressed levels in in it was not possible to A2 gene in L. major by this analysis a these L. major A2 genes are not or are not the A2 genes in L. major are not and this with the in that the remaining A2 genes in major represent of the few in A2 in L. major and the of A2 on L. major in and in detailed are differences in the A2 gene structure and expression between L. donovani and L. major. it was that the A2 regulatory sequence was in L. donovani and L. major. It has been that the sequence was responsible for amastigote-specific expression in L. donovani H. Zhang W.W. Matlashewski G. J. Biol. Chem. 1996; 271: 17081-17090Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and that it was possible to express A2 in L. major (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar). It was therefore to the A2 sequence the ability to amastigote-specific expression in L. major. L. major promastigotes with the A2-expressing which a of the L. donovani A2 gene the A2 protein coding and the regulatory as in with a control with no A2 coding analysis was carried out to A2 expression be amastigote in the L. major in it was possible to A2 expression by the to the sequence of L. major A2 has >90% to A2 in L. donovani, suggesting that the L. major A2 and associated regulatory functional in L. major with respect to amastigote-specific Although it has been that expression of A2 the ability of L. major to in the spleen of BALB/c mice after intravenous infection (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar), it was to study of A2-expressing L. major. It was A2 expression the growth of L. major in or in in macrophages. in L. major with the as the control L. major A2-expressing and the control L. major infected and in macrophages after infection with parasite and these demonstrate that it was possible to amastigote-specific gene expression of A2 in L. major, and this not the ability to in or in in macrophages. of A2-expressing L. major in BALB/c major infections cutaneous and the gene structure and revealed that L. major the ability to express the A2 therefore an to A2 expression the of cutaneous leishmaniasis by L. major in infection The and control L. major promastigotes into the of BALB/c mice, and the infection was by the for to the first the A2-expressing L. major swelling of the levels to the control infected mice after this the swelling in the A2 was in Moreover, in the mice infected with the control L. major on after but no of lesions in mice infected with the A2-expressing L. major. of from the infected after infection revealed that the mice infected with control L. major to than infected with the A2-expressing L. major. These observations that expression of A2 protein the ability of L. major amastigotes to establish cutaneous infections. The carried with L. major a of the A2 are copies of A2 genes in L. proteins ranging from to 100 depending on the of sequences each gene (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar, 10Zhang W.W. Charest H. Ghedin E. Matlashewski G. Mol. Biochem. Parasitol. 1996; 78: 79-90Crossref PubMed Scopus (122) Google Scholar). It was therefore to the L. major of copies of the A2 in analysis of L. major with the A2 containing revealed the of A2 proteins of and which expressed specifically further the that the A2 is functional in L. major with respect to amastigote-specific Although the A2 and control L. major in macrophages than the they have in infection in macrophages and in not We therefore to BALB/c mice with control and A2 L. major, and the was followed as in the A2 L. major to swelling and in no detectable infection with the control containing L. major, which was with respect to cutaneous The on cutaneous infection was with the A2 L. major than with the A2 L. major, suggesting that multiple copies of A2 protein the of cutaneous infection than expression of a of the A2 It is that out the that genes on this to the We that the genes are repeated with the A2 genes as in and are present on this not the A2 phenotype A2 and into L. major not of A2-expressing L. major in C57BL/6 carried out on the of genetically susceptible BALB/c mice and that restoring A2 expression to L. major in suppression of cutaneous It to further this phenotype in an infection that closely the pathogenesis of human cutaneous leishmaniasis S. G. J. N. E. J. J. PubMed Scopus Google Scholar). We therefore the of A2 expression on the cutaneous pathogenesis by L. major infection with L. major promastigotes after in in genetically resistant C57BL/6 mice. was by the of the the of the infection. in of C57BL/6 mice infected with the control L. major lesions as as after after and spontaneously after this the typical L. major infection in resistant C57BL/6 mice S. G. J. N. E. J. J. PubMed Scopus Google Scholar). with was in the infections in susceptible BALB/c mice, the A2 L. major to in C57BL/6 mice Moreover, parasite in and in A2-expressing L. major infected mice than in control L. mice and The of from mice infected with A2-expressing L. major after infection was to that the lack of growth of the A2-expressing L. major in of infected C57BL/6 mice. These that A2 expression in L. major cutaneous It was to examine the immune response the A2-expressing L. major to the suppression of survival in the skin was attributable to an acquired immune response against A2-expressing L. major or attributable to an of A2 on L. major in the skin from of C57BL/6 mice infected for with control or A2 L. major and with or L. The after and for and in the control L. major infection in C57BL/6 mice expression of and expression of a immune detected in the of mice infected with A2-expressing L. major, suggesting that the host immune response was not in the infection with A2-expressing L. major. These argue that the suppression of cutaneous infection was attributable to an of A2 on L. major survival in the with A2-expressing L. major of the L. inability of A2-expressing L. major to in cutaneous or of mice is of L. donovani. It was therefore of to A2 expression in L. major of L. donovani. We have a to the ability of macrophages infected with different species of Leishmania to out of the macrophages with a and infected with different of L. major or L. donovani are in the of C57BL/6 mice, and are and the from the are and by these that macrophages infected with species out of the in higher levels than macrophages infected with cutaneous species not It was therefore possible to infected with A2-expressing L. major L. macrophages in these in the of L. macrophages out of the was than macrophages infected with control L. major. infected with A2-expressing L. major out of the skin in a higher than infected with the control L. major These results that A2-expressing L. major L. donovani with respect to ability to and from the skin in the study of Leishmania is species cutaneous leishmaniasis the site of or no cutaneous pathology but from the site of infection and fatal visceral infections. to this is through differences between Leishmania species and to genetically engineered to express species-specific this major observations have been in the present a detailed analysis of the gene expression, and gene of the A2 in L. donovani and L. major demonstrate that although L. major A2 gene sequences, the A2 protein coding repeated sequences are absent in the L. major A2 genes and are no it was possible to restore amastigote-specific A2 expression to L. major, and this the ability of L. major to establish cutaneous infections in resistant or susceptible mice. L. major is closely related but to have and from L. donovani PubMed Scopus Google Scholar, Kenney R.T. Mol. Biochem. Parasitol. 1996; PubMed Scopus Google Scholar). these observations suggest that or of A2 expression in Old World Leishmania a in the species-specific tropism in Leishmania infection. The inability of A2-expressing L. major to cutaneous infection in mice was an to L. donovani, which levels of A2 in amastigotes and is to cutaneous lesions or to in the skin in mice or humans. We to macrophages infected with L. donovani, macrophages infected with A2-expressing L. major an ability to out of the with the control L. macrophages. A2-expressing L. major to establish a infection in the skin or the of is likely to for the that was no detectable cellular immune response against the A2-expressing L. major in the C57BL/6 mice, a response against the control L. major. A2 appeared to an suppression of L. major survival in the It has been that A2 expression the ability of L. major to in the resident macrophages of the in in BALB/c mice after infection through injection (8Zhang W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar). We have results in the A. and G. A2 expression in L. major has different of infection in resident macrophages of visceral organs but infection in macrophages in the A2 expression in Leishmania therefore have in different of macrophages. to differences between control and A2-expressing L. major in infection of resident or macrophages in the genetic it was to that although L. major does not express A2 proteins, they to be A2 pseudogenes. The A2 genes in L. major are specifically in the protein coding and are not expressed into these genes the that between L. donovani and L. major. Moreover, L. major the ability to amastigote-specific expression from the A2 the regions of the A2 genes in L. donovani and L. major are and than the coding In coding sequences are than regions is a against the gene Although it is not possible to L. donovani A2 genes or L. major A2 sequences the that the A2 was from L. major because the A2 regulatory sequences and the A2 protein coding sequence are present in the genome of L. major. The L. donovani A2 protein coding regions are present in Leishmania species associated with visceral leishmaniasis L. donovani, L. and L. but not in cutaneous leishmaniasis species as L. major, L. and L. (11Ghedin E. Zhang W.W. Charest H. Sundar S. Kenney R.T. Matlashewski G. Clin. Diagn. Lab. Immunol. 1997; 4: 530-535Crossref PubMed Google Scholar), and this with the that A2 a in visceral the A2 coding sequences detected by karyotype in L. L. and L. (11Ghedin E. Zhang W.W. Charest H. Sundar S. Kenney R.T. Matlashewski G. Clin. Diagn. Lab. Immunol. 1997; 4: 530-535Crossref PubMed Google Scholar), and the A2 gene have been detected by analysis as proteins of kDa in L. from the World L. are associated with cutaneous infections, and they are from the Old World species L. donovani and L. major PubMed Scopus Google Scholar). There therefore be differences in the of the genome that L. to in the skin the of the A2 in gene between Old World and World Leishmania species not be because although is a virulence factor for L. major L. B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar), it not to be a virulence factor for L. J. PubMed Scopus Google Scholar). differences be related to the in the host response to different species of Leishmania as Trends Parasitol. 2001; Full Text Full Text PDF Scopus (140) Google Scholar). The different of A2 proteins be associated with the different and of these proteins because A2 proteins are in L. than in L. It is that has been of visceral Leishmania infections by L. a cutaneous Leishmania species N. J. PubMed Scopus Google Scholar, Kenney R.T. Lancet. PubMed Google Scholar). Although the host and immune in the outcome of Leishmania infection Kenney R.T. Lancet. PubMed Google Scholar), it is not known the of these L. tropica These observations the argument that A2 is not the factor required for visceral in the of L. donovani, it does appear to be for visceral infection (7Zhang W.W. Matlashewski G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8807-8811Crossref PubMed Scopus (134) Google Scholar, W.W. Matlashewski G. Mol. Microbiol. 2001; 39: 935-948Crossref PubMed Scopus (98) Google Scholar). the Leishmania genome E. S. A. S. A. Biochem. PubMed Scopus Google Scholar, A. J. A. U. S. Biol. Sci. PubMed Scopus Google Scholar), it be of to differences between Leishmania species that a of the tropism and pathogenesis of these

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