Invasion Of Mammalian Cells Research Articles

The PagN protein of Salmonella enterica serovar Typhimurium is an adhesin and invasin

BackgroundThe pagN gene of Salmonella enterica serovar Typhimurium is a PhoP-regulated gene that is up-regulated during growth within macrophages and in vivo in murine models of infection. The PagN protein displays similarity to the Hek and Tia invasins/adhesins of Escherichia coli. Thus far no function has been ascribed to the PagN protein.ResultsHere we show that the outer membrane located PagN protein mediates agglutination of red blood cells and that this can be masked by LPS. When expressed in Escherichia coli the PagN protein supports adhesion to and invasion of mammalian cells in a manner that is dependent on cytoskeletal rearrangements. S. enterica sv Typhimurium pagN mutants display a reduction in adhesion to and invasion of epithelial cells. Finally, we demonstrate that over-expression of PagN in a SPI-1 mutant can partially compensate for the lack of a functional invasasome.ConclusionPagN is an outer membrane protein that may contribute to the virulence of S. Typhimurium. This protein is a haemagglutinin and contributes to the adherence to mammalian cells. In addition, PagN can mediate high-level invasion of CHO-K1 cells. Previously,pagN mutants have been shown to be less competitive in vivo and thus this may be due to their lessened ability to interact with mammalian cells. Finally PagN can be added to an ever-growing repertoire of factors that contribute to the pathogenesis of Salmonella.

Adhesin-dependent binding and uptake of Salmonella enterica serovar Typhimurium by dendritic cells

Salmonella enterica serovar Typhimurium can be internalized by immature dendritic cells (DCs). The interacting host and bacterial molecules initiating this process remain uncharacterized. The objective of this study was to investigate whether specific fimbriae are involved in the early step of binding and uptake of Salmonella by DCs. Type 1 fimbriated S. enterica serovar Typhimurium or recombinant Escherichia coli expressing the type 1 fimbriae showed a significantly greater ability to attach to murine bone-marrow-derived DCs than non-fimbriated bacteria. The FimH adhesin was required for efficient interactions with DCs, since fimbriated fimH mutants were impaired in both binding and internalization. Finally, the internalization involved a FimH-dependent process but did not require sipB, a gene essential for Salmonella-mediated invasion of mammalian epithelial cells. Collectively, these data suggest that the bacterial interaction of DCs through the type 1 fimbrial adhesin FimH is sufficient to target S. enterica serovar Typhimurium for cellular uptake.

The Alternative Sigma Factor σBand the Virulence Gene Regulator PrfA Both Regulate Transcription ofListeria monocytogenesInternalins

Some Listeria monocytogenes internalins are recognized as contributing to invasion of mammalian tissue culture cells. While PrfA is well established as a positive regulator of L. monocytogenes virulence gene expression, the stress-responsive sigma(B) has been recognized only recently as contributing to expression of virulence genes, including some that encode internalins. To measure the relative contributions of PrfA and sigma(B) to internalin gene transcription, we used reverse transcription-PCR to quantify transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG) in L. monocytogenes 10403S and in isogenic Delta prfA, Delta sigB, and Delta sigB Delta prfA strains. Strains were grown under defined conditions to produce (i) active PrfA, (ii) active sigma(B) and active PrfA, (iii) inactive PrfA, and (iv) active sigma(B) and inactive PrfA. Under the conditions tested, sigma(B) and PrfA contributed differentially to the expression of the various internalins such that (i) both sigma(B) and PrfA contributed to inlA and inlB transcription, (ii) only PrfA contributed to inlC transcription, (iii) only sigma(B) contributed to inlC2 and inlD transcription, and (iv) neither sigma(B) nor PrfA contributed to inlF and inlG transcription. inlE transcript levels were undetectable. The important role for sigma(B) in regulating expression of L. monocytogenes internalins suggests that exposure of this organism to environmental stress conditions, such as those encountered in the gastrointestinal tract, may activate internalin transcription. Interplay between sigma(B) and PrfA also appears to be critical for regulating transcription of some virulence genes, including inlA, inlB, and prfA.

Paxillin phosphorylation: bifurcation point downstream of integrin-linked kinase (ILK) in streptococcal invasion

Efficient group A streptococcus (GAS) invasion of mammalian cells requires fibronectin (Fn) binding proteins, such as M1 and PrtF1/SfbI, that bridge bacteria to integrins and activate cellular signalling for ingestion. Previous studies of GAS invasion, mediated by both proteins, suggest a common signalling pathway. However, distinct cellular morphological changes at the port of bacterial entry suggest that different signals are also induced. Here we report that paxillin is phosphorylated in response to Fn-bound GAS that express either M1 or PrtF1/SfbI protein, but is not phosphorylated in response to a mutant deficient in both proteins. Inhibition of paxillin phosphorylation by a tyrosine kinase inhibitor, PP2, or by expression of a dominant negative form of paxillin significantly reduced invasion by M1+ but did not affect ingestion of PrtF1/SfbI+ strains. In contrast, another tyrosine inhibitor, genistein, did not significantly prevent paxillin phosphorylation and had no effect on ingestion of the M1+ strain, but reduced PrtF1/SfbI-mediated entry. This suggests that paxillin phosphorylation is induced by both proteins but only required for M1-mediated invasion. A bifurcation point, downstream of integrin-linked kinase (ILK) and phosphoinositide 3-kinase, likely accounts for the distinct morphological changes. Furthermore, ILK activity is indispensable for M1-induced paxillin recruitment and phosphorylation.

Environmental signal integration by a modular AND gate

Microorganisms use genetic circuits to integrate environmental information. We have constructed a synthetic AND gate in the bacterium Escherichia coli that integrates information from two promoters as inputs and activates a promoter output only when both input promoters are transcriptionally active. The integration occurs via an interaction between an mRNA and tRNA. The first promoter controls the transcription of a T7 RNA polymerase gene with two internal amber stop codons blocking translation. The second promoter controls the amber suppressor tRNA supD. When both components are transcribed, T7 RNA polymerase is synthesized and this in turn activates a T7 promoter. Because inputs and outputs are promoters, the design is modular; that is, it can be reconnected to integrate different input signals and the output can be used to drive different cellular responses. We demonstrate this modularity by wiring the gate to integrate natural promoters (responding to Mg2+ and AI-1) and using it to implement a phenotypic output (invasion of mammalian cells). A mathematical model of the transfer function is derived and parameterized using experimental data.

The Trypanosoma cruzi metacyclic-specific protein Met-III associates with the nucleolus and contains independent amino and carboxyl terminal targeting elements

Metacyclogenesis in Trypanosoma cruzi involves the differentiation of replicating non-infective epimastigotes into non-replicating metacyclic trypomastigotes. This pre-adapts parasites for infection of the mammalian host and is characterised by several morphological changes and structural alterations to the nucleus, including nucleolar disaggregation. Experimental investigation of these developmental processes has been hampered by a lack of robust molecular markers. Here, we describe the precise temporal expression of the T. cruzi-specific protein Met-III, in the genome reference strain CL Brener. Expression is restricted to metacyclics in the insect stages of the life-cycle and is rapidly down-regulated following invasion of mammalian cells. Met-III localises to dispersed foci typical of the disassembled nucleolus in metacyclics and to the discrete single nucleolus of cells soon after macrophage invasion. To identify elements that target Met-III, we generated a series of tagged green fluorescent protein fusion proteins and examined their sub-nuclear location in transformed parasites. These experiments demonstrated that amino and carboxyl terminal fragments, characterised by clusters of basic residues, could independently mediate nucleolar sequestration. To investigate the function of Met-III, we used gene deletion. This showed that Met-III is not required for the development of metacyclic trypomastigotes and that null mutants can complete the life-cycle in vitro.

High-throughput assays of phagocytosis, phagosome maturation, and bacterial invasion

Ingestion of foreign particles by macrophages and neutrophils and the fate of the vacuole that contains the ingested material are generally monitored by optical microscopy. Invasion of host cells by pathogenic bacteria and their intracellular proliferation are similarly studied by microscopy or by plating assays. These labor-intensive and time-consuming methods limit the number of assays that can be performed. The effort required to test multiple reagents or conditions can be prohibitive. We describe high-throughput assays of phagocytosis and of phagosomal maturation. An automated fluorescence microscope-based platform and associated analysis software were used to study Fcgamma receptor-mediated phagocytosis of IgG-opsonized particles by cultured murine macrophages. Phagosomal acidification was measured as an index of maturation. The same platform was similarly used to implement high-throughput assays of invasion of mammalian cells by pathogenic bacteria. The invasion of HeLa cells by Salmonella and the subsequent intracellular proliferation of the bacteria were measured rapidly and reliably in large populations of cells. These high-throughput methods are ideally suited for the efficient screening of chemical libraries to select potential drugs and of small interference RNA libraries to identify essential molecules involved in critical steps of the immune response.

Down-Regulation of Key Virulence Factors Makes the Salmonella enterica Serovar Typhimurium rfaH Mutant a Promising Live-Attenuated Vaccine Candidate

Mutants of Salmonella enterica serovar Typhimurium that lack the transcriptional regulator RfaH are efficient as live oral vaccines against salmonellosis in mice. We show that the attenuation of the vaccine candidate strain is associated with reduced net growth in epithelial and macrophage cells. In order to identify the relevant RfaH-dependent genes, the RfaH regulon was determined with S. enterica serovars Enteritidis and Typhimurium using whole-genome Salmonella microarrays. As well as impacting the expression of genes involved in lipopolysaccharide (LPS) core and O-antigen synthesis, the loss of RfaH results in a marked down-regulation of SPI-4 genes, the flagellum/chemotaxis system, and type III secretion system 1. However, a proportion of these effects could have been the indirect consequence of the altered expression of genes required for LPS biosynthesis. Direct and indirect effects of the rfaH mutation were dissociated by genome-wide transcriptional profiling of a structural deep-rough LPS mutant (waaG). We show that truncation of LPS itself is responsible for the decreased intracellular yield observed for DeltarfaH strains. LPS mutants do not differ in replication ability; rather, they show increased susceptibility to antimicrobial peptides in the intracellular milieu. On the other hand, evidence that deletion of rfaH, as well as some other genes involved in LPS biosynthesis, results in enhanced invasion of various mammalian cells is shown. Exposure of common minor antigens in the absence of serovar-specific antigens might be responsible for the observed cross-reactive nature of the elicited immune response upon vaccination. Increased invasiveness of the Salmonella rfaH mutant into antigen-presenting cells, combined with increased intracellular killing and the potential for raising a cross-protective immune response, renders the rfaH mutant an ideal vaccine candidate.

Involvement of Ssp-4-related carbohydrate epitopes in mammalian cell invasion by Trypanosoma cruzi amastigotes

We examined whether the expression of Ssp-4-related carbohydrate epitopes defined by monoclonal antibodies 1D9 and 2B7 was related to cell invasion by Trypanosoma cruzi amastigotes from different isolates and whether the highest expression of the epitope defined by MAb 1D9 would confer greater infectivity. Confocal microscopy showed that both epitopes localize to the membrane of amastigotes from 569, 588, 573, 587 and SC2005 isolates, similar to the G isolate, whereas the CL isolate showed a punctate and diffuse staining. Flow cytometry revealed inter- and intra-isolate variable expression of these epitopes. Apart from the lower expression of MAb 2B7 epitope by intracellular amastigotes of the SC2005 isolate, amastigotes from chagasic patient isolates expressed both epitopes similar to the G isolate, in contrast to CL isolate, that showed lower expression of both epitopes. MAb 1D9 did not react with CL isolate on immunoblots and reacted poorly with 588 and 587 parasites. MAb 2B7 preferentially reacted with an epitope on an 84kDa component in G and 573 isolates. Invasion assays revealed that despite the fact that amastigotes from chagasic patient isolates displayed high levels of the epitope defined by MAb 1D9, only isolate 588 invaded host cells in levels comparable to that of isolate G. Both MAbs specifically inhibited cell invasion by G and 588, but not CL. These results suggested that the highest expression of MAb 1D9 epitope was not sufficient to confer higher infectivity on the isolate, and besides the two epitopes, other factors may modulate the invasiveness of extracellular amastigotes from the different isolates.

Localization of the C3-Like ADP-Ribosyltransferase from Staphylococcus aureus during Bacterial Invasion of Mammalian Cells

The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase which possesses no specific receptor-binding domain or translocation unit required for entry in target cells where its substrate is located. Here we show that C3stau2 can reach its target after invasion of staphylococci in eukaryotic cells without needing translocation.

Molecular basis of mammalian cell invasion by Trypanosoma cruzi

Establishment of infection by Trypanosoma cruzi, the agent of Chagas' disease, depends on a series of events involving interactions of diverse parasite molecules with host components. Here we focus on the mechanisms of target cell invasion by metacyclic trypomastigotes (MT) and mammalian tissue culture trypomastigotes (TCT). During MT or TCT internalization, signal transduction pathways are activated both in the parasite and the target cell, leading to Ca2+ mobilization. For cell adhesion, MT engage surface glycoproteins, such as gp82 and gp35/50, which are Ca2+ signal-inducing molecules. In T. cruzi isolates that enter host cells in gp82-mediated manner, parasite protein tyrosine kinase as well as phospholipase C are activated, and Ca2+ is released from I P3-sensitive stores, whereas in T. cruzi isolates that attach to target cells mainly through gp35/50, the signaling pathway involving adenylate cyclase appears to be stimulated, with Ca2+ release from acidocalciosomes. In addition, T. cruzi isolate-dependent inhibitory signals, mediated by MT-specific gp90, may be triggered both in the host cell and the parasite. The repertoire of TCT molecules implicated in cell invasion includes surface glycoproteins of gp85 family, with members containing binding sites for laminin and cytokeratin 18, enzymes such as cruzipain, trans-sialidase, and an oligopeptidase B that generates a Ca2+-agonist from a precursor molecule.

Molecular Characterization of Serine-, Alanine-, and Proline-Rich Proteins of Trypanosoma cruzi and Their Possible Role in Host Cell Infection

We previously reported the isolation of a novel protein gene family, termed SAP (serine-, alanine-, and proline-rich protein), from Trypanosoma cruzi. Aided by the availability of the completed genome sequence of T. cruzi, we have now identified 39 full-length sequences of SAP, six pseudogenes and four partial genes. SAPs share a central domain of about 55 amino acids and can be divided into four groups based on their amino (N)- and carboxy (C)-terminal sequences. Some SAPs have conserved N- and C-terminal domains encoding a signal peptide and a glycosylphosphatidylinositol anchor addition site, respectively. Analysis of the expression of SAPs in metacyclic trypomastigotes by two-dimensional electrophoresis and immunoblotting revealed that they are likely to be posttranslationally modified in vivo. We have also demonstrated that some SAPs are shed into the extracellular medium. The recombinant SAP exhibited an adhesive capacity toward mammalian cells, where binding was dose dependent and saturable, indicating a possible ligand-receptor interaction. SAP triggered the host cell Ca2+ response required for parasite internalization. A cell invasion assay performed in the presence of SAP showed inhibition of internalization of the metacyclic forms of the CL strain. Taken together, these results show that SAP is involved in the invasion of mammalian cells by metacyclic trypomastigotes, and they confirm the hypothesis that infective trypomastigotes exploit an arsenal of surface glycoproteins and shed proteins to induce signaling events required for their internalization.

Streptococcal modulation of cellular invasion via TGF-β1 signaling

Group A Streptococcus (GAS) and other bacterial pathogens are known to interact with integrins as an initial step in a complex pathway of bacterial ingestion by host cells. Efficient GAS invasion depends on the interaction of bound fibronectin (Fn) with integrins and activation of integrin signaling. TGF-beta1 regulates expression of integrins, Fn, and other extracellular matrix proteins, and positively controls the integrin signaling pathway. Therefore, we postulated that TGF-beta1 levels could influence streptococcal invasion of mammalian cells. Pretreatment of HEp-2 cells with TGF-beta1 increased their capacity to ingest GAS when the bacteria expressed fibronectin-binding proteins (M1 or PrtF1). Western blots revealed significant induction of alpha5 integrin and Fn expression by HEp-2 cells in response to TGF-beta1. Increased ingestion of streptococci by these cells was blocked by a specific inhibitor of the TGF-beta1 receptor I and antibodies directed against alpha5 integrin and Fn, indicating that increased invasion depends on TGF-beta1 up-regulation of both the alpha5 integrin and Fn. The capacity of TGF-beta1 to up-regulate integrin expression and intracellular invasion by GAS was reproduced in primary human tonsil fibroblasts, which could be a source of TGF-beta1 in chronically infected tonsils. The relationship between TGF-beta1 and GAS invasion was strengthened by the observation that TGF-beta1 production was stimulated in GAS-infected primary human tonsil fibroblasts. These findings suggest a mechanism by which GAS induce a cascade of changes in mammalian tissue leading to elevated expression of the alpha5beta1 receptor, enhanced invasion, and increased opportunity for survival and persistence in their human host.

Solution Structure and Backbone Dynamics of the Trypanosoma cruzi Cysteine Protease Inhibitor Chagasin

A Trypanosoma cruzi cysteine protease inhibitor, termed chagasin, is the first characterized member of a new family of tight-binding cysteine protease inhibitors identified in several lower eukaryotes and prokaryotes but not present in mammals. In the protozoan parasite T.cruzi, chagasin plays a role in parasite differentiation and in mammalian host cell invasion, due to its ability to modulate the endogenous activity of cruzipain, a lysosomal-like cysteine protease. In the present work, we determined the solution structure of chagasin and studied its backbone dynamics by NMR techniques. Structured as a single immunoglobulin-like domain in solution, chagasin exerts its inhibitory activity on cruzipain through conserved residues placed in three loops in the same side of the structure. One of these three loops, L4, predicted to be of variable length among chagasin homologues, is flexible in solution as determined by measurements of (15)N relaxation. The biological implications of structural homology between chagasin and other members of the immunoglobulin super-family are discussed.

Proteinases of Trypanosoma cruzi: Potential Targets for the Chemotherapy of Chagas Disease

Trypanosoma cruzi, the agent of the American Trypanosomiasis, Chagas Disease, contains cysteine, serine, threonine and metallo proteinases. Aspartic proteinases have not been found so far. The most abundant among these enzymes is cruzipain, a cysteine proteinase expressed as a complex mixture of isoforms by the major developmental stages of the parasite, including some membrane-bound isoforms. The enzyme is an immunodominant antigen in human chronic Chagas disease and seems to be important in the host/parasite relationship. Inhibitors of cruzipain kill the parasite and cure infected mice, thus making the enzyme a very promising target for the development of new drugs against Chagas disease. In addition 30 kDa cathepsin B-like enzymes have been described. Serine peptidases described in the parasite include oligopeptidase B, a member of the prolyl oligopeptidase family involved in Ca(2+)-signalling during mammalian cell invasion; a prolyl endopeptidase (Tc80), against which inhibitors are being developed, and a serine carboxypeptidase belonging to the S10 family. Metalloproteinases homologous to the gp63 of Leishmania spp. are also present. The proteasome has properties similar to those of other eukaryotes, and its inhibition by lactacystin blocks some differentiation steps in the life cycle of the parasite.

Cloning and characterization of the phosphoglucomutase of Trypanosoma cruzi and functional complementation of a Saccharomyces cerevisiae PGM null mutant

Trypanosoma cruzi is the etiological agent of Chagas' disease, a chronic illness characterized by progressive cardiomyopathy and/or denervation of the digestive tract. The parasite surface is covered with glycoconjugates, such as mucin-type glycoproteins and glycoinositolphospholipids (GIPLs), whose glycans are rich in galactopyranose (Galp) and/or galactofuranose (Galf) residues. These molecules have been implicated in attachment of the parasite to and invasion of mammalian cells and in modulation of the host immune responses during infection. In T. cruzi, galactose (Gal) biosynthesis depends on the conversion of uridine diphosphate (UDP)-glucose (UDP-Glc) into UDP-Gal by an NAD-dependent reduction catalyzed by UDP-Gal 4-epimerase. Phosphoglucomutase (PGM) is a key enzyme in this metabolic pathway catalyzing the interconversion of Glc-6-phosphate (Glc-6-P) and Glc-1-P which is then converted into UDP-Glc. We here report the cloning of T. cruzi PGM, encoding T. cruzi PGM, and the heterologous expression of a functional enzyme in Saccharomyces cerevisiae. T. cruzi PGM is a single copy gene encoding a predicted protein sharing 61% amino acid identity with Leishmania major PGM and 43% with the yeast enzyme. The 59-trans-splicing site of PGM RNA was mapped to a region located at 18 base pairs upstream of the start codon. Expression of T. cruzi PGM in a S. cerevisiae null mutant-lacking genes encoding both isoforms of PGM (pgm1Delta/pgm2Delta) rescued the lethal phenotype induced upon cell growth on Gal as sole carbon source.

Regulation of Tissue-Specific and Extracellular Matrix-Related Genes by a Class I Histone Deacetylase

Class I histone deacetylases (HDACs) repress transcription by deacetylating histones and have been shown to play crucial roles in mouse, Xenopus, zebrafish, and C. elegans development. To identify the molecular networks regulated by a class I HDAC in a multicellular organism, we carried out a global gene expression profiling study using C. elegans embryos, and identified tissue-specific and extracellular matrix (ECM)-related genes as major HDA-1 targets. Ectopic expression of HDA-1 or C. elegans cystatin, an HDA-1 target identified from the microarray, significantly perturbed mammalian cell invasion. Similarly, RNAi depletion or overexpression of human HDAC-1 also affected cell migration. These findings suggest that HDA-1/HDAC-1 may play a critical, evolutionarily conserved role in regulating the extracellular microenvironment. Because human HDACs are targets for cancer therapy, these findings have significant implications in cancer treatment.

The TibA adhesin/invasin from enterotoxigenic Escherichia coli is self recognizing and induces bacterial aggregation and biofilm formation.

Escherichia coli strains are responsible for many cases of gastrointestinal disease and represent a serious health problem worldwide. An essential step in the pathogenesis of such strains involves recognition and attachment to host intestinal surfaces. TibA is a potent bacterial adhesin associated with a number of enterotoxigenic E. coli strains and mediates bacterial attachment to a variety of human cells; additionally, it promotes invasion of such cells. This adhesin is a surface-displayed autotransporter protein and belongs to the exclusive group of bacterial glycoproteins; only the glycosylated form confers binding to and invasion of mammalian cells. Here we characterized TibA and showed that it possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrated that intercellular TibA-TibA interaction is responsible for bacterial autoaggregation. Also, TibA expression significantly enhances biofilm formation by E. coli on abiotic surfaces.

Chagasin, the endogenous cysteine-protease inhibitor ofTrypanosoma cruzi, modulates parasite differentiation and invasion of mammalian cells

Chagasin is a Trypanosoma cruzi protein that was recently characterized as a tight-binding inhibitor of papain-like cysteine proteases (CPs). Considering that parasite virulence and morphogenesis depend on the endogenous activity of lysosomal CPs of the cruzipain family, we sought to determine whether chagasin and cruzipain interact in the living cell. Ultrastructural studies showed that chagasin and cruzipain both localize to the Golgi complex and reservosomes (lysosome-like organelles), whereas free chagasin was found in small intracellular vesicles, suggesting that chagasin trafficking pathways might intersect with those of cruzipain. Taking advantage of the fact that sodium dodecyl sulphate and beta-mercaptoethanol prevent binding between the isolated proteins but do not dismantle preformed cruzipain-chagasin complexes, we obtained direct evidence that chagasin-cruzipain complexes are indeed formed in epimastigotes. Chagasin transfectants (fourfold increase in CP inhibitory activity) displayed low rates of differentiation (metacyclogenesis) and exhibited increased resistance to a synthetic CP inhibitor. These phenotypic changes were accompanied by a drastic reduction of soluble cruzipain activity and by upregulated secretion of cruzipain-chagasin molecular complexes. Analysis of six T. cruzi strains revealed that expression levels of cruzipain and chagasin are variable, but the molar ratios are fairly stable ( approximately 50:1) in most strains, with the exception of the G strain (5:1), which is poorly infective. On the same vein, we found that trypomastigotes overexpressing chagasin are less infective than wild-type parasites in vitro. The deficiency of chagasin overexpressers is caused by lower activity of membrane-associated CPs, because membranes recovered from wild-type trypomastigotes restored infectivity and this effect was nullified by the CP inhibitor E-64. In summary, our studies suggest that chagasin regulates the endogenous activity of CP, thus indirectly modulating proteolytic functions that are essential for parasite differentiation and invasion of mammalian cells.

Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

Trypanosoma cruzi, the etiological agent of Chagas disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.