Mechanism of C. trachomatis attachment to eukaryotic host cells.

A novel trimolecular mechanism of microbial attachment to mammalian host cells was characterized for the obligate intracellular pathogen Chlamydia trachomatis. Using purified glycosaminoglycans (GAGs) and specific GAG lyases, we demonstrated that a heparan sulfate-like GAG present on the surface of chlamydia organisms is required for attachment to host cells. These observations were supported by inhibition of attachment following binding of heparan sulfate receptor analogs to chlamydiae and by demonstrating that chlamydiae synthesize a unique heparan sulfate-like GAG. Furthermore, exogenous heparan sulfate, as an adhesin analog, restored attachment and infectivity to organisms that had lost these attributes following treatment with heparan sulfate lyase. These data suggest that a GAG adhesin ligand mediates attachment by bridging mutual GAG receptors on the host cell surface and on the chlamydial outer membrane surface.     

Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.     

Inhibitory effect of heparan sulfate-like glycosaminoglycans on the infectivity of Chlamydia pneumoniae in HL cells varies between strains

Glycosaminoglycans are known to participate in the attachment of several chlamydial strains. We studied the effect of heparin, enoxaparin, low-molecular-weight heparin, chondroitin sulfate A, and heparinase I on the infectivity of Chlamydia pneumoniae strain CWL029 and two Finnish isolates, Kajaani 7 and Parola, in an HL cell line which is epithelial in origin. Two Chlamydia trachomatis strains, L2 and E, were used for comparison. The infectivity of all C. pneumoniae strains and C. trachomatis serovar E was inhibited not only by heparin derivatives but also by chondroitin sulfate A and heparinase treatment. Treatment of host cells with heparin derivatives and heparinase was also inhibitory. Different chlamydial strains and species seem, however, to vary in their ability to use heparin in their attachment to host cells.     

α2-3- and α2-6- N-linked sialic acids allow efficient interaction of Newcastle Disease Virus with target cells

Receptor recognition and binding is the first step in the viral cycle. It has been established that Newcastle Disease Virus (NDV) interacts with sialylated molecules such as gangliosides and glycoproteins at the cell surface. Nevertheless, the specific receptor(s) that mediate virus entry are not well known. We have analysed the role of the sialic acid linkage in the early steps of the viral infection cycle. Pretreatment of ELL-0 cells with both α2,3 and α2,6 specific sialidases led to the inhibition of NDV binding, fusion and infectivity, which were restored after α2,3(N)- and α2,6(N)-sialyltransferase incubation. Moreover, α2,6(N)-sialyltransferases also restored NDV activities in α2-6-linked sialic acid deficient cells. Competition with α2-6 sialic acid-binding lectins led to a reduction in the three NDV activities (binding, fusion and infectivity) suggesting a role for α2-6- linked sialic acid in NDV entry. We conclude that both α2-3- and α2-6- linked sialic acid containing glycoconjugates may be used for NDV infection. NDV was able to efficiently bind, fuse and infect the ganglioside-deficient cell line GM95 to a similar extent to that of its parental MEB4, suggesting that gangliosides are not essential for NDV binding, fusion and infectivity. Nevertheless, the fact that the interaction of NDV with cells deficient in N-glycoprotein expression such as Lec1 was less efficient prompted us to conclude that NDV requires N-linked glycoproteins for efficient attachment and entry into the host cell.     

VLA-2 (α2β1) Integrin Promotes Rotavirus Entry into Cells but Is Not Necessary for Rotavirus Attachment

In an attempt to identify the rotavirus receptor, we tested 46 cell lines of different species and tissue origins for susceptibility to infection by three N-acetyl-neuraminic (sialic) acid (SA)-dependent and five SA-independent rotavirus strains. Susceptibility to SA-dependent or SA-independent rotavirus infection varied depending on the cell line tested and the multiplicity of infection (MOI) used. Cells of renal or intestinal origin and transformed cell lines derived from breast, stomach, bone, or lung were all susceptible to rotavirus infection, indicating a wider host tissue range than previously appreciated. Chinese hamster ovary (CHO), baby hamster kidney (BHK-21), guinea pig colon (GPC-16), rat small intestine (Rie1), and mouse duodenum (MODE-K) cells were found to support only limited rotavirus replication even at MOIs of 100 or 500, but delivery of rotavirus particles into the cytoplasm by lipofection resulted in efficient rotavirus replication. The rotavirus cell attachment protein, the outer capsid spike protein VP4, contains the sequence GDE(A) recognized by the VLA-2 (alpha2beta1) integrin, and to test if VLA-2 is involved in rotavirus attachment and entry, we measured infection in CHO cells that lack VLA-2 and CHO cells transfected with the human alpha2 subunit (CHOalpha2) or with both the human alpha2 and beta1 subunits (CHOalpha2beta1) of VLA-2. Infection by SA-dependent or SA-independent rotavirus strains was 2- to 10-fold more productive in VLA-2-expressing CHO cells than in parental CHO cells, and the increased susceptibility to infection was blocked with anti-VLA-2 antibody. However, the levels of binding of rotavirus to CHO, CHOalpha2, and CHOalpha2beta1 cells were equivalent and were not increased over binding to susceptible monkey kidney (MA104) cells or human colonic adenocarcinoma (Caco-2, HT-29, and T-84) cells, and binding was not blocked by antibody to the human alpha2 subunit. Although the VLA-2 integrin promotes rotavirus infection in CHO cells, it is clear that the VLA-2 integrin alone is not responsible for rotavirus cell attachment and entry. Therefore, VLA-2 is not involved in the initial attachment of rotavirus to cells but may play a role at a postattachment level.     

Rotavirus VP4 and VP7-Derived Synthetic Peptides as Potential Substrates of Protein Disulfide Isomerase Lead to Inhibition of Rotavirus Infection

Rotavirus infection of MA104 cells has been shown to be inhibited by cell membrane-impermeant thiol/disulfide exchange inhibitors and anti-PDI antibodies. To characterise the amino acid sequences of rotavirus structural proteins potentially mediating cell surface PDI?Csubstrate interactions, rotavirus-derived peptides from VP4 and VP7 (RRV) and VP7 (Wa), and their modified versions containing serine instead of cysteine were synthesized. Cysteine-containing VP7 peptides corresponding to residues 189?C210 or 243?C263 caused an infectivity inhibitory effect of about 64 and 85?%, respectively, when added to cells. Changing cysteine to serine significantly decreased the inhibitory effect. A cysteine-containing peptide corresponding to VP4 residues 200?C219 and its scrambled version reduced infectivity by 92 and 80?%, respectively. A cysteine to serine change in the original VP4 200?C219 peptide did not affect its inhibitory effect. Non-rotavirus related sequences containing cysteine residues efficiently inhibited rotavirus infectivity. Antibodies against VP7 residues 189?C210 or 243?C263 significantly inhibited rotavirus infectivity only after virus attachment to cells had occurred, whereas those against VP4 200?C219 peptide inhibited infectivity irrespective of whether virus or cell-attached virus was antibody-treated. A direct PDI?Cpeptide interaction was shown by ELISA for cysteine-containing VP7 and VP4 peptides. Virus?Ccell attachment was unaffected by the peptides inhibiting virus infectivity. The results showed that even though cysteine residues in the peptides tested are important in both virus infectivity inhibition and in vitro PDI?Cpeptide interaction, the accompanying amino acid sequence also plays some role. As a whole, our findings further support our hypothesis that cell surface PDI from MA104 cells might be contributing to rotavirus entry at a post-attachment step.     

Epithelial membrane protein 2 modulates infectivity of Chlamydia muridarum (MoPn)

Chlamydiae are bacterial pathogens which have evolved efficient strategies to enter, replicate, and survive inside host epithelial cells, resulting in acute and chronic diseases in humans and other animals. Several candidate molecules in the host receptor complex have been identified, but the precise mechanisms of infection have not been elucidated. Epithelial membrane protein-2 (EMP2), a 4-transmembrane protein, is highly expressed in epithelial cells in sites of chlamydial infections. Here we show that infectivity of the Chlamydia muridarum (MoPn) is associated with host cellular expression of EMP2 in multiple cell lines. Recombinant knockdown of EMP2 impairs infectivity, whereas infectivity is augmented in cells recombinantly modified to over-express EMP2. An epithelial cell line without native expression of EMP2 is relatively resistant to MoPn infection, whereas infectivity is markedly increased by recombinant expression of EMP2 in that cell line. Blockade of surface EMP2 using a specific anti-EMP2 antibody significantly reduces chlamydial infection efficiency. In addition, MoPn infectivity as measured in the EMP2 overexpressing cell line is not heparin-dependent, suggesting a possible role for EMP2 in the non-reversible phase of early infection. These findings identify EMP2 as a candidate host protein involved in infection of C. muridarum (MoPn).     

Internalization and processing of Bacillus anthracis lethal toxin by toxin-sensitive and -resistant cells

Anthrax lethal toxin consists of two separate proteins, protective antigen and lethal factor (LF). Certain macrophages and a mouse macrophage-like cell line, J774A.1, are lysed by low concentrations of lethal toxin. In contrast, another macrophage cell line, IC-21, and all other cell types tested were resistant to this toxin. To discover the basis for this difference, each step in the intoxication process was examined. No differences between sensitive and resistant cells were found in receptor binding or proteolytic activation of protective antigen, steps that are required prior to LF binding. To determine whether resistance results from a defect in translocation to the cytosol, we introduced LF into J774A.1 and IC-21 cells and a nonmacrophage cell line (L6 myoblast) by osmotic lysis of pinocytic vesicles. Only J774A.1 cells were lysed; no effect was observed in IC-21 and L6 cells. These results suggest that resistant cells either lack the intracellular target of LF or fail to process LF to an active form. The relatively low potency of LF introduced into J774A.1 cells by osmotic lysis suggests that protective antigen may also be required at a stage subsequent to endocytosis.     

The soluble serum protein Gas6 bridges virion envelope phosphatidylserine to the TAM receptor tyrosine kinase Axl to mediate viral entry

Virus entry into cells is typically initiated by binding of virally encoded envelope proteins to specific cell surface receptors. Studying infectivity of lentivirus pseudotypes lacking envelope binding, we still observed high infectivity for some cell types. On further investigation, we discovered that this infectivity is conferred by the soluble bovine protein S in fetal calf serum, or Gas6, its human homolog. Gas6 enhances native infectivity of pseudotypes of multiple viral envelope proteins. Gas6 mediates binding of the virus to target cells by bridging virion envelope phosphatidylserine to Axl, a TAM receptor tyrosine kinase on target cells. Phagocytic clearance of apoptotic cells is known to involve bridging by Gas6. Replication of vaccinia virus, which was previously reported to use apoptotic mimicry to enter cells, is also enhanced by Gas6. These results reveal an alternative molecular mechanism of viral entry that can broaden host range and enhance infectivity of enveloped viruses.     

Proteolysis of the Ebola virus glycoproteins enhances virus binding and infectivity

Cellular cathepsins are required for Ebola virus infection and are believed to proteolytically process the Ebola virus glycoprotein (GP) during entry. However, the significance of cathepsin cleavage during infection remains unclear. Here we demonstrate a role for cathepsin L (CatL) cleavage of Ebola virus GP in the generation of a stable 18-kDa GP1 viral intermediate that exhibits increased binding to and infectivity for susceptible cell targets. Cell binding to a lymphocyte line was increased when CatL-proteolysed pseudovirions were used, but lymphocytes remained resistant to Ebola virus GP-mediated infection. Genetic removal of the highly glycosylated mucin domain in Ebola virus GP resulted in cell binding similar to that observed with CatL-treated full-length GP, and no overall enhancement of binding or infectivity was observed when mucin-deleted virions were treated with CatL. These results suggest that cathepsin cleavage of Ebola virus GP facilitates an interaction with a cellular receptor(s) and that removal of the mucin domain may facilitate receptor binding. The influence of CatL in Ebola virus GP receptor binding should be useful in future studies characterizing the mechanism of Ebola virus entry.     

Recent insights into the mechanisms of Chlamydia entry

Chlamydia are widespread bacteria that grow in human and animal cells. They enter their host cell, establish an intracellular environment favourable for their multiplication and finally exit the host cell. A combination of host cell factors and of bacterial proteins contribute to pathogen entry. Recent advances have shed new light on the entry mechanism, following attachment. Here we review recent data concerning endocytosis, host cell signalling, proteins secreted by the bacteria, the actin cytoskeleton in entry and the involvement of small GTPases.     

N-linked glycosylation facilitates sialic acid-independent attachment and entry of influenza A viruses into cells expressing DC-SIGN or L-SIGN

It is widely recognized that sialic acid (SA) can mediate attachment of influenza virus to the cell surface, and yet the specific receptors that mediate virus entry are not known. For many viruses, a definitive demonstration of receptor function has been achieved when nonpermissive cells are rendered susceptible to infection following transfection of the gene encoding a putative receptor. For influenza virus, such approaches have been confounded by the abundance of SA on mammalian cells so that it has been difficult to identify cell lines that are not susceptible to infection. We examined influenza virus infection of Lec2 Chinese hamster ovary (CHO) cells, a mutant cell line deficient in SA. Lec2 CHO cells were resistant to influenza virus infection, and stable cell lines expressing either DC-SIGN or L-SIGN were generated to assess the potential of each molecule to function as SA-independent receptors for influenza A viruses. Virus strain BJx109 (H3N2) bound to Lec2 CHO cells expressing DC-SIGN or L-SIGN in a Ca(2+)-dependent manner, and transfected cells were susceptible to virus infection. Treatment of Lec2-DC-SIGN and Lec2-L-SIGN cells with mannan, but not bacterial neuraminidase, blocked infection, a finding consistent with SA-independent virus attachment and entry. Moreover, virus strain PR8 (H1N1) bears low levels of mannose-rich glycans and was inefficient at infecting Lec2 CHO cells expressing either DC-SIGN or L-SIGN, whereas other glycosylated H1N1 subtype viruses could infect cells efficiently. Together, these data indicate that human C-type lectins (DC-SIGN and L-SIGN) can mediate attachment and entry of influenza viruses independently of cell surface SA.     

Terminal N-linked galactose is the primary receptor for adeno-associated virus 9

Sialylated glycans serve as cell surface attachment factors for a broad range of pathogens. We report an atypical example, where desialylation increases cell surface binding and infectivity of adeno-associated virus (AAV) serotype 9, a human parvovirus isolate. Enzymatic removal of sialic acid, but not heparan sulfate or chondroitin sulfate, increased AAV9 transduction regardless of cell type. Viral binding and transduction assays on mutant Chinese hamster ovary (CHO) cell lines defective in various stages of glycan chain synthesis revealed a potential role for core glycan residues under sialic acid in AAV9 transduction. Treatment with chemical inhibitors of glycosylation and competitive inhibition studies with different lectins suggest that N-linked glycans with terminal galactosyl residues facilitate cell surface binding and transduction by AAV9. In corollary, resialylation of galactosylated glycans on the sialic acid-deficient CHO Lec2 cell line with different sialyltransferases partially blocked AAV9 transduction. Quantitative analysis of AAV9 binding to parental, sialidase-treated or sialic acid-deficient mutant CHO cells revealed a 3-15-fold increase in relative binding potential of AAV9 particles upon desialylation. Finally, pretreatment of well differentiated human airway epithelial cultures and intranasal instillation of recombinant sialidase in murine airways enhanced transduction efficiency of AAV9 by >1 order of magnitude. Taken together, the studies described herein provide a molecular basis for low infectivity of AAV9 in vitro and a biochemical strategy to enhance gene transfer by AAV9 vectors in general.     

Methylamine facilitates demonstration of specific uptake of diphtheria toxin by CHO cell and toxin-resistant CHO cell mutants

We have measured the specific uptake of ¹²⁵I-labelled diphtheria toxin in the presence of methylamine by a number of cell lines with different sensitivities to diphtheria toxin. The results show a strong correlation between the toxin sensitivities of the cell lines and the amount of specific uptake. The specific association of labelled toxin with cells was clearly demonstrated even with CHO cells, a cell line with relatively low sensitivity. Thus, CHO cell mutants that are resistant to diphtheria toxin could be classified as toxin-binding or non-binding cells by this method.     

Effect of increased expression of protein disulfide isomerase and heavy chain binding protein on antibody secretion in a recombinant CHO cell line

Previous work has shown that a human-antibody-producing recombinant CHO cell line did not increase its intracellular content of protein disulfide isomerase (PDI) and heavy chain binding protein (BIP) according to the increasing expression of antibody. It was also found that the intracellular assembly of light and heavy chain is a major limiting factor for overall cell specific productivity, as secretion rates improve with higher light chain expression levels and heavy chain accumulates intracellularly when too little light chain is present. As these CHO cells had a significantly lower intracellular PDI content compared to that of hybridoma cells, these results have led us to try to overcome the limitation in the posttranslational assembly in the endoplasmatic reticulum. Recombinant CHO cells were transfected with PDI or BIP alone or in combination, and the effect on intracellular light and heavy chain content and specific production rate was determined. Overexpression of BIP, both alone and in combination with PDI, reduced the specific secretion rate, whereas PDI, when overexpressed alone, caused an increase of product secretion rate.     

Specific binding sites for a parvovirus, minute virus of mice, on cultured mouse cells.

The early interactions between parvoviruses and host cells have not been extensively described previously. In this study we have characterized some aspects of viral binding to the cell surface and demonstrated the existence of specific cellular receptor sites for minute virus of mice (MVM) on two murine cell lines that are permissive for viral growth. The interaction had a pH optimum of 7.0 to 7.2, and both the rate and extent of the reactions were slightly affected by temperature. Mouse A-9 cells (L-cell derivative) had approximately 5 X 10(5) specific MVM binding sites per cell, and Friend erythroleukemia cells had 1.5 X 10(5) MVM sites per cell. In contrast, the nonpermissive mouse lymphoid cell line L1210 lacked specific viral receptors. Also, cloned lines of A-9 cells resistant to viral infection have been isolated. One of these lines lacked the "specific" virus attachment sites but exhibited low levels of nonsaturable virus binding. Based on these examples, infectivity is correlated with the presence of specific viral receptors on the cell surface.     

The Chlamydia outer membrane protein OmcB is required for adhesion and exhibits biovar-specific differences in glycosaminoglycan binding

Chlamydia pneumoniae, an obligate intracellular human pathogen, causes a number of respiratory diseases. We explored the role of the conserved OmcB protein in C. pneumoniae infections, using yeast display technology. (i) Yeast cells presenting OmcB were found to adhere to human epithelial cells. (ii) Pre-incubation of OmcB yeast cells with heparin, but not other glycosaminoglycans (GAGs), abrogated adhesion. (iii) Pre-treatment of the target cells with heparinase inhibited adherence, and GAG-deficient CHO cell lines failed to bind OmcB yeast. (iv) A heparin-binding motif present near the N-terminus of OmcB is required for host cell binding. (v) Pre-treatment of chlamydial elementary bodies (EBs) with anti-OmcB antibody or pre-incubation of target cells with recombinant OmcB protein reduced infectivity upon challenge with C. pneumoniae. (vi) Adhesion of fluorescently labelled EBs to epithelial or endothelial cells was abrogated by prior addition of heparin or OmcB protein. Thus, C. pneumoniae OmcB is an adhesin that binds heparan sulphate-like GAGs. OmcB from Chlamydia trachomatis serovar L1 also adheres to human cells in a heparin-dependent way, unlike its counterpart from serovar E. We show that a single position in the OmcB sequence determines heparin dependence/independence, and variations there may reflect differences between the two serovars in cell tropism and disease pattern.     

An early event in the herpes simplex virus type-2 replication cycle is sufficient to induce Chlamydia trachomatis persistence

Epidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Transmission electron microscopic (TEM) analyses demonstrated that in both HeLa and HEC-1B cells, co-infection caused developing chlamydiae to exhibit swollen, aberrantly shaped reticulate bodies (RBs), characteristically observed in persistence. Additionally, HSV-2 co-infection suppressed production of infectious chlamydial elementary bodies (EBs) in both host cell types. Co-infection with HSV type 1 (HSV-1) produced similar morphologic alterations and abrogated infectious EB production. These data indicate that virus-induced chlamydial persistence was neither host cell- nor virus strain-specific. Purification of crude HSV-2 stocks demonstrated that viral particles were required for coinfection-induced chlamydial persistence to occur. Finally, co-infection with either UV-inactivated, replication-incompetent virus or replication-competent HSV-2 in the presence of cyclohexamide reduced chlamydial infectivity without altering chlamydial genomic DNA accumulation. These data demonstrate that productive viral replication is not required for the induction of chlamydial persistence and suggest that HSV attachment and entry can provide the necessary stimulus to alter C. trachomatis development.     

Adenovirus interaction with distinct integrins mediates separate events in cell entry and gene delivery to hematopoietic cells.

A major impediment to the effective use of adenovirus vectors for gene therapy is a lack of knowledge of how these vectors interact with diverse cell types in vivo. Adenovirus attachment to most human cell types is mediated by the fiber protein, which binds to an as yet unidentified cell receptor. In contrast to this, we report that adenovirus type 2 (Ad2) attachment to hematopoietic cells is facilitated by interaction of the penton base protein with members of the beta2 integrin family. Adenovirus particles were capable of binding to human monocytic cells, which lack fiber receptors, and virus binding could be blocked by a soluble penton base or by a function-blocking monoclonal antibody to integrin alphaMbeta2. To confirm the role of alphaMbeta2 integrins in Ad2 binding to hematopoietic cells, we analyzed virus attachment and gene delivery to CHO cells expressing recombinant beta2 integrins. alphaMbeta2-expressing CHO cells supported 3- to 5-fold-higher levels of Ad2 binding and 5- to 10-fold-larger amounts of gene delivery than did nontransfected CHO cells, indicating that alphaMbeta2 facilitates adenovirus attachment to and infection of hematopoietic cells. While beta2 integrins promote Ad2 attachment to hematopoietic cells, further studies demonstrated that alphav integrins were required for the next step in infection, virus internalization into cell endosomes. These studies reveal a novel pathway of Ad2 infection of hematopoietic cells mediated by distinct integrins which facilitate separate events in virus entry. They also suggest a possible strategy for selective adenovirus-mediated gene delivery to hematopoietic cells.     

Monoclonal antibody of IgG isotype against a cross-reactive lipopolysaccharide epitope of Chlamydia and Salmonella Re chemotype enhances infectivity in L-929 fibroblast cells

A murine monoclonal antibody (MAb) 202D7 of IgG3 isotype recognizes a lipopolysaccharide (LPS) epitope of Chlamydia spp. and cross-reacts with the Re chemotype LPS of Salmonella and Escherichia coli. The antibody exhibits strong complement activating properties and stimulates phagocytosis of Salmonella enterica serovar Minnesota Re mutant by murine macrophages. Salmonella Re mutants are non-invasive for cell monolayers but still can enter and replicate in L-929 murine fibroblast cells. The entry of bacteria within the cells increases five-fold in the presence of MAb 202D7. The antibody mediates attachment and enhances five-fold the infectivity of Chlamydia pneumoniae into L-929 cells, which suggests a possible IgG-mediated mechanism of entry and survival of the pathogen in fibroblast cells.