Leishmania regulates host macrophage miRNAs expression by engaging transcription factor c-Myc.

Parasites of Leishmania genus have developed sophisticated strategies allowing them to deactivate their host macrophage to promote their survival. It has become clear that miRNAs play important roles in shaping innate and adaptive immune responses toward pathogens. It is not surprising that several pathogens including Leishmania have evolved the ability to regulate host macrophage miRNA expression in order to manipulate host cell phenotypes to their advantage. However, very little is known about the mechanisms used by intracellular pathogens to drive changes in host cell miRNA abundance. In this review, Leishmania exploitation of macrophage transcription factor c-Myc as a critical proxy virulence factor to regulate abundance of macrophage miRNAs influencing macrophage physiology to promote its survival will be discussed.

Miransertib (ARQ 092), an orally-available, selective Akt inhibitor is effective against Leishmania.

Leishmaniasis is amongst the most important neglected diseases, afflicting more than 12 million people in 88 countries. There is an urgent need for safe orally bioavailable and cost-effective drugs for the treatment of leishmaniasis. It has recently been shown that Leishmania activates host macrophage serine/threonine kinase Akt, to promote survival of both parasites and infected cells. Here, we sought to evaluate a compound, Miransertib (ARQ 092), an orally bioavailable and selective allosteric Akt inhibitor currently in clinical trials for patients with PI3K/Akt-driven tumors or Proteus syndrome. Miransertib was tested against Leishmania donovani and Leishmania amazonensis, causative agents of visceral and cutaneous leishmaniasis, respectively. Cultured promastigotes were susceptible to Miransertib. In addition, Miransertib was markedly effective against intracellular amastigotes of L. donovani or L. amazonensis-infected macrophages. Miransertib also enhanced mTOR dependent autophagy in Leishmania-infected macrophages, which may represent one mechanism of Miransertib-mediated killing of intracellular Leishmania. Whereas parasite clearance in the spleen of mice infected with L. donovani and treated with Miransertib was comparable to that when treated with miltefosine, Miransertib caused a greater reduction in the parasite load in the liver. In the cutaneous leishmaniasis infection model, lesions were reduced by 40% as compared to mock treated mice. Together, these results provide direct evidence to support the conclusion that Miransertib is an excellent lead compound for the development of a new oral drug therapy for visceral and cutaneous leishmaniasis.

c-Myc is a novel Leishmania virulence factor by proxy that targets the host miRNA system and is essential for survival in human macrophages.

Leishmania species are intracellular protozoan pathogens that have evolved to successfully infect and deactivate host macrophages. How this deactivation is brought about is not completely understood. Recently, microRNAs (miRNAs) have emerged as ubiquitous regulators of macrophage gene expression that contribute to shaping the immune responses to intracellular pathogens. Conversely, several pathogens have evolved the ability to exploit host miRNA expression to manipulate host-cell phenotype. However, very little is known about the mechanisms used by intracellular pathogens to drive changes in host-cell miRNA abundance. Using miRNA expression profiling of Leishmania donovani-infected human macrophages, we show here that Leishmania infection induced a genome-wide down-regulation of host miRNAs. This repression occurred at the level of miRNA gene transcription, because the synthesis rates of primary miRNAs were significantly decreased in infected cells. miRNA repression depended on the host macrophage transcription factor c-Myc. Indeed, the expression of host c-Myc was markedly up-regulated by Leishmania infection, and c-Myc silencing reversed the miRNA suppression. Furthermore, c-Myc silencing significantly reduced intracellular survival of Leishmania, demonstrating that c-Myc is essential for Leishmania pathogenesis. Taken together, these findings identify c-Myc not only as being responsible for miRNA repression in Leishmania-infected macrophages but also as a novel and essential virulence factor by proxy that promotes Leishmania survival.

Countervailing, time-dependent effects on host autophagy promotes intracellular survival of Leishmania.

Autophagy is essential for cell survival under stress and has also been implicated in host defense. Here, we investigated the interactions between Leishmania donovani, the main etiological agent of visceral leishmaniasis, and the autophagic machinery of human macrophages. Our results revealed that during early infection-and via activation of the Akt pathway-Leishmania actively inhibits the induction of autophagy. However, by 24 h, Leishmania switched from being an inhibitor to an overall inducer of autophagy. These findings of a dynamic, biphasic response were based on the accumulation of lipidated light chain 3 (LC3), an autophagosome marker, by Western blotting and confocal fluorescence microscopy. We also present evidence that Leishmania induces delayed host cell autophagy via a mechanism independent of reduced activity of the mechanistic target of rapamycin (mTOR). Notably, Leishmania actively inhibited mTOR-regulated autophagy even at later stages of infection, whereas there was a clear induction of autophagy via some other mechanism. In this context, we examined host inositol monophosphatase (IMPase), reduced levels of which have been implicated in mTOR-independent autophagy, and we found that IMPase activity is significantly decreased in infected cells. These findings indicate that Leishmania uses an alternative pathway to mTOR to induce autophagy in host macrophages. Finally, RNAi-mediated down-regulation of host autophagy protein 5 (ATG5) or autophagy protein 9A (ATG9A) decreased parasite loads, demonstrating that autophagy is essential for Leishmania survival. We conclude that Leishmania uses an alternative pathway to induce host autophagy while simultaneously inhibiting mTOR-regulated autophagy to fine-tune the timing and magnitude of this process and to optimize parasite survival.

Leishmania donovani chaperonin 10 regulates parasite internalization and intracellular survival in human macrophages.

Protozoa of the genus Leishmania infect macrophages in their mammalian hosts causing a spectrum of diseases known as the leishmaniases. The search for leishmania effectors that support macrophage infection is a focus of significant interest. One such candidate is leishmania chaperonin 10 (CPN10) which is secreted in exosomes and may have immunosuppressive properties. Here, we report for the first time that leishmania CPN10 localizes to the cytosol of infected macrophages. Next, we generated two genetically modified strains of Leishmania donovani (Ld): one strain overexpressing CPN10 (CPN10+++) and the second, a CPN10 single allele knockdown (CPN10+/-), as the null mutant was lethal. When compared with the wild-type (WT) parental strain, CPN10+/- Ld showed higher infection rates and parasite loads in human macrophages after 24 h of infection. Conversely, CPN10+++ Ld was associated with lower initial infection rates. This unexpected apparent gain-of-function for the knockdown could have been explained either by enhanced parasite internalization or by enhanced intracellular survival. Paradoxically, we found that CPN10+/- leishmania were more readily internalized than WT Ld, but also displayed significantly impaired intracellular survival. This suggests that leishmania CPN10 negatively regulates the rate of parasite uptake by macrophages while being required for intracellular survival. Finally, quantitative proteomics identified an array of leishmania proteins whose expression was positively regulated by CPN10. In contrast, many macrophage proteins involved in innate immunity were negatively regulated by CPN10. Taken together, these findings identify leishmania CPN10 as a novel effector with broad based effects on macrophage cell regulation and parasite survival.

Mp1p Is a Virulence Factor in Talaromyces (Penicillium) marneffei.

BACKGROUND: Talaromyces marneffei is an opportunistic dimorphic fungus prevalent in Southeast Asia. We previously demonstrated that Mp1p is an immunogenic surface and secretory mannoprotein of T. marneffei. Since Mp1p is a surface protein that can generate protective immunity, we hypothesized that Mp1p and/or its homologs are virulence factors. METHODOLOGY/PRINCIPAL FINDINGS: We examined the pathogenic roles of Mp1p and its homologs in a mouse model. All mice died 21 and 30 days after challenge with wild-type T. marneffei PM1 and MP1 complemented mutant respectively. None of the mice died 60 days after challenge with MP1 knockout mutant (P<0.0001). Seventy percent of mice died 60 days after challenge with MP1 knockdown mutant (P<0.0001). All mice died after challenge with MPLP1 to MPLP13 knockdown mutants, suggesting that only Mp1p plays a significant role in virulence. The mean fungal loads of PM1 and MP1 complemented mutant in the liver, lung, kidney and spleen were significantly higher than those of the MP1 knockout mutant. Similarly, the mean load of PM1 in the liver, lung and spleen were significantly higher than that of the MP1 knockdown mutant. Histopathological studies showed an abundance of yeast in the kidney, spleen, liver and lung with more marked hepatic and splenic necrosis in mice challenged with PM1 compared to MP1 knockout and MP1 knockdown mutants. Likewise, a higher abundance of yeast was observed in the liver and spleen of mice challenged with MP1 complemented mutant compared to MP1 knockout mutant. PM1 and MP1 complemented mutant survived significantly better than MP1 knockout mutant in macrophages at 48 hours (P<0.01) post-infection. The mean fungal counts of Pichia pastoris GS115-MP1 in the liver (P<0.001) and spleen (P<0.05) of mice were significantly higher than those of GS115 at 24 hours post-challenge. CONCLUSIONS/SIGNIFICANCE: Mp1p is a key virulence factor of T. marneffei. Mp1p mediates virulence by improving the survival of T. marneffei in macrophages.

Phenotypic Characterization of a Leishmania donovani Cyclophilin 40 Null Mutant.

Protozoan parasites of the genus Leishmania adapt to their arthropod and vertebrate hosts through the development of defined life cycle stages. Stage differentiation is triggered by environmental stress factors and has been linked to parasite chaperone activities. Using a null mutant approach we previously revealed important, nonredundant functions of the cochaperone cyclophilin 40 in L. donovani-infected macrophages. Here, we characterized in more detail the virulence defect of cyp40-/- null mutants. In vitro viability assays, infection tests using macrophages, and mixed infection experiments ruled out a defect of cyp40-/- parasites in resistance to oxidative and hydrolytic stresses encountered inside the host cell phagolysosome. Investigation of the CyP40-dependent proteome by quantitative 2D-DiGE analysis revealed up regulation of various stress proteins in the null mutant, presumably a response to compensate for the lack of CyP40. Applying transmission electron microscopy we showed accumulation of vesicular structures in the flagellar pocket of cyp40-/- parasites that we related to a significant increase in exosome production, a phenomenon previously linked to the parasite stress response. Together these data suggest that cyp40-/- parasites experience important intrinsic homeostatic stress that likely abrogates parasite viability during intracellular infection.

Synergistic effects of diosmetin with erythromycin against ABC transporter over-expressed methicillin-resistant Staphylococcus aureus (MRSA) RN4220/pUL5054 and inhibition of MRSA pyruvate kinase.

Increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) worldwide with limited therapeutic options is a growing public health concern. Natural products have been shown to possess antibacterial actions against MRSA. Flavonoids from natural products have been shown to possess antibacterial actions against MRSA by antagonizing its resistance mechanisms. Diosmin and diosmetin are natural flavonoids found in a variety of citrus fruits. The aim of this study was to investigate whether diosmin and diosmetin could inhibit the growth of MRSA and the in vitro enzymatic activity of a newly discovered MRSA drug target, pyruvate kinase (PK). By using a panel of MRSA strains with known resistant mechanisms, neither diosmin nor diosmetin was shown to possess direct antibacterial activities against all tested MRSA strains. However, in checkerboard assay, we found that diosmetin together with erythromycin, could synergistically inhibit the growth of ABC-pump overexpressed MRSA-RN4220/pUL5054, and time kill assay also showed that the antibacterial activities of diosmetin with erythromycin were bactericidal. Diosmetin was further shown to significantly suppress the MRSA PK activities in a dose dependent manner. In conclusion, the inhibition of MRSA PK by diosmetin could lead to a deficiency of ATP and affect the bacterial efflux pump which might contribute to the antibacterial actions of diosmetin against MRSA.

Identification of the Mycobacterium tuberculosis protein PE-PGRS62 as a novel effector that functions to block phagosome maturation and inhibit iNOS expression.

Using a genetic screen in yeast we found that Mycobacterium tuberculosis PE-PGRS62 was capable of disrupting yeast vacuolar protein sorting, suggesting effects on endosomal trafficking. To study the impact of PE-PGRS62 on macrophage function, we infected murine macrophages with Mycobacterium smegmatis expressing PE-PGRS62. Infected cells displayed phagosome maturation arrest. Phagosomes acquired Rab5, but displayed a significant defect in Rab7 and LAMP-1 acquisition. Macrophages infected with M. smegmatis expressing PE-PGRS62 also expressed two- to threefold less iNOS protein when compared with cells infected with wild-type bacteria. Consistent with this, cells infected with a Mycobacterium marinum transposon mutant for the PE-PGRS62 orthologue showed greater iNOS protein expression when compared to cells infected with wild-type organisms. Complementation restored the ability of the mutant to inhibit iNOS expression. No differences in iNOS transcript levels were observed, suggesting that PE-PGRS62 effects on iNOS expression occurred post-transcriptionally. Marked differences in colony morphology were also seen in M. smegmatis expressing PE-PGRS62 and in the M. marinum transposon mutant, suggesting that PE-PGRS62 may affect cell wall composition. These findings suggest that PE-PGRS62 supports virulence via inhibition of phagosome maturation and iNOS expression, and these phenotypes may be linked to effects on bacterial cell wall composition.

Class IA phosphatidylinositol 3-kinase p110α regulates phagosome maturation.

Of the various phosphatidylinositol 3- kinases (PI3Ks), only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5)P3; however, p110α and PI(3,4,5)P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, ß-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP) and homotypic vacuole fusion and protein sorting (HOPs) components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K.

Secreted virulence factors and immune evasion in visceral leishmaniasis.

Evasion or subversion of host immune responses is a well-established paradigm in infection with visceralizing leishmania. In this review, we summarize current findings supporting a model in which leishmania target host regulatory molecules and pathways, such as the PTP SHP-1 and the PI3K/Akt signaling cascade, to prevent effective macrophage activation. Furthermore, we describe how virulence factors, secreted by leishmania, interfere with macrophage intracellular signaling. Finally, we discuss mechanisms of secretion and provide evidence that leishmania use a remarkably adept, exosome-based secretion mechanism to export and deliver effector molecules to host cells. In addition to representing a novel mechanism for trafficking of virulence factors across membranes, recent findings indicate that leishmania exosomes may have potential as vaccine candidates.

Antibacterial activity, inflammatory response, coagulation and cytotoxicity effects of silver nanoparticles.

The incorporation of nanoparticles (NPs) in industrial and biomedical applications has increased significantly in recent years, yet their hazardous and toxic effects have not been studied extensively. Here, we studied the effects of 24 nm silver NPs (AgNPs) on a panel of bacteria isolated from medical devices used in a hospital intensive care unit. The cytotoxic effects were evaluated in macrophages and the expression of the inflammatory cytokines IL-6, IL-10 and TNF-α were quantified. The effects of NPs on coagulation were tested in vitro in plasma-based assays. We demonstrated that 24 nm AgNPs were effective in suppressing the growth of clinically relevant bacteria with moderate to high levels of antibiotic resistance. The NPs had a moderate inhibitory effect when coagulation was initiated through the intrinsic pathway. However, these NPs are cytotoxic to macrophages and are able to elicit an inflammatory response. Thus, beneficial and potential harmful effects of 24 nm AgNPs on biomedical devices must be weighed in further studies in vivo. From the Clinical Editor: The authors of this study demonstrate that gallic acid reduced 24 nm Ag NPs are effective in suppressing growth of clinically relevant antibiotic resistant bacteria. However, these NPs also exhibit cytotoxic properties to macrophages and may trigger an inflammatory response. Thus, the balance of beneficial and potential harmful effects must be weighed carefully in further studies.

Myeloid cell IL-10 production in response to leishmania involves inactivation of glycogen synthase kinase-3ß downstream of phosphatidylinositol-3 kinase.

Leishmania disease expression has been linked to IL-10. In this study, we investigated the regulation of IL-10 production by macrophages infected with Leishmania donovani. Infection of either murine or human macrophages brought about selective phosphorylation of Akt-2 in a PI3K-dependent manner. These events were linked to phosphorylation and inactivation of glycogen synthase kinase-3ß (GSK-3ß) at serine 9, as the latter was abrogated by inhibition of either PI3K or Akt. One of the transcription factors that is negatively regulated by GSK-3ß is CREB, which itself positively regulates IL-10 expression. Infection of macrophages with leishmania induced phosphorylation of CREB at serine 133, and this was associated with enhanced CREB DNA binding activity and induction of IL-10. Similar to phosphorylation of GSK-3ß, both phosphorylation of CREB at serine 133 and CREB DNA binding activity were abrogated in cells treated with inhibitors of either PI3K or Akt prior to infection. Furthermore, disruption of this pathway either by inhibition of Akt or by overexpression of GSK-3ß markedly attenuated IL-10 production in response to leishmania. Thus, GSK-3ß negatively regulates myeloid cell IL-10 production in response to leishmania. Switching off GSK-3ß promotes disease pathogenesis.

An exosome-based secretion pathway is responsible for protein export from Leishmania and communication with macrophages.

Specialized secretion systems are used by numerous bacterial pathogens to export virulence factors into host target cells. Leishmania and other eukaryotic intracellular pathogens also deliver effector proteins into host cells; however, the mechanisms involved have remained elusive. In this report, we identify exosome-based secretion as a general mechanism for protein secretion by Leishmania, and show that exosomes are involved in the delivery of proteins into host target cells. Comparative quantitative proteomics unambiguously identified 329 proteins in Leishmania exosomes, accounting for >52% of global protein secretion from these organisms. Our findings demonstrate that infection-like stressors (37 degrees C +/- pH 5.5) upregulated exosome release more than twofold and also modified exosome protein composition. Leishmania exosomes and exosomal proteins were detected in the cytosolic compartment of infected macrophages and incubation of macrophages with exosomes selectively induced secretion of IL-8, but not TNF-alpha. We thus provide evidence for an apparently broad-based mechanism of protein export by Leishmania. Moreover, we describe a mechanism for the direct delivery of Leishmania molecules into macrophages. These findings suggest that, like mammalian exosomes, Leishmania exosomes function in long-range communication and immune modulation.

Myeloid cell differentiation in response to calcitriol for expression CD11b and CD14 is regulated by myeloid zinc finger-1 protein downstream of phosphatidylinositol 3-kinase.

Immature cells of the mononuclear phagocyte series differentiate in response to calcitriol. This is accompanied by increased expression of both CD11b and CD14 and has been shown to be phosphatidylinositol 3-kinase (PI3K) dependent. The events downstream of PI3K that regulate mononuclear phagocyte gene expression, however, remain to be fully understood. In the present study, we show that incubation of THP-1 cells with calcitriol brings about activation of the myeloid zinc finger-1 (MZF-1) transcription factor dependent upon PI3K. In addition, we show that the proximal promoter regions of both CD11b and CD14 contain functional MZF-1 binding sites that are calcitriol responsive. Site-directed mutagenesis of the putative MZF-1 elements abolished MZF-1 binding to the promoters of both CD11b and CD14. Not only did calcitriol treatment increase MZF-1 DNA binding activity to these sites, but it also up-regulated cellular levels of MZF-1. Silencing of MZF-1 resulted in a markedly blunted response to calcitriol for induction of both CD11b and CD14 mRNA transcript levels. Cell surface expression of CD11b and CD14 was also reduced, but to a lesser extent. Taken together, these results show that MZF-1 is involved downstream of PI3K in a calcitriol-induced signaling pathway leading to myeloid cell differentiation and activation of CD11b and CD14.

The vitamin D receptor-mediated activation of phosphatidylinositol 3-kinase (PI3Kalpha) plays a role in the 1alpha,25-dihydroxyvitamin D3-stimulated increase in steroid sulphatase activity in myeloid leukaemic cell lines.

In this article we show that 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) stimulates the activity of the class IA phosphatidylinositol 3-kinase PI3Kalpha and its downstream target Akt in HL60, U937 and THP-1 myeloid leukaemic cell lines. Furthermore, we show that the classical nuclear vitamin D receptor (VDR(nuc)) is involved in this activation of the PI3K/Akt signalling in these cell lines. We have previously shown that the activity of steroid sulphatase is stimulated in HL60, U937 and THP-1 myeloid leukaemic cell lines by 1alpha,25(OH)(2)D(3) (Hughes et al., [2001] Biochem J 355:361-371; Hughes et al., [2005] J Cell Biochem 94:1175-1189; Hughes and Brown [2006] J Cell Biochem 98:590-617). In this article we show that the 1alpha,25(OH)(2)D(3)-stimulated increase in signalling via the PI3K/Akt pathway plays a role in the increase in steroid sulphatase activity in the HL60 U937 and THP-1 cell lines. We used a variety of pharmacological and biochemical approaches to show that activation of PI3Kalpha mediates the 1alpha,25(OH)(2)D(3)-stimulated increase in steroid sulphatase activity in myeloid leukaemic cells. We also show that the PI3K/Akt dependent activation of NF-kappaB plays a role in the 1alpha,25(OH)(2)D(3)-stimulated increase in steroid sulphatase activity in myeloid leukaemic cells.

Annexin V associates with the IFN-gamma receptor and regulates IFN-gamma signaling.

Many of the biological activities of IFN-gamma are mediated through the IFN-gammaR3-linked Jak-Stat1alpha pathway. However, regulation of IFN-gamma signaling is not fully understood, and not all responses to IFN-gamma are Stat1alpha dependent. To identify novel elements involved in IFN-gamma cell regulation, the cytoplasmic domain of the R2 subunit of the human IFN-gammaR was used as bait in a yeast two-hybrid screen of a human monocyte cDNA library. This identified annexin A5 (AxV) as a putative IFN-gammaR binding protein. The interaction was confirmed in pull-down experiments in which a GST-R2 cytoplasmic domain fusion protein was incubated with macrophage lysates. Furthermore, immunoprecipitation using anti-IFN-gammaR2 Abs showed that AxV interacted with IFN-gammaR2 to form a stable complex following incubation of cells with IFN-gamma. In 293T cells with reduced expression of AxV, brought about by small interfering RNA targeting, activation of Jak2 and Stat1alpha in response to IFN-gamma was enhanced. Inhibition of cell proliferation, a hallmark of the IFN-gamma response, also was potentiated in HeLa cells treated with small interfering RNA directed at AxV. Taken together, these results suggest that through an inducible association with the R2 subunit of the IFN-gammaR, AxV modulates cellular responses to IFN-gamma by modulating signaling through the Jak-Stat1 pathway.

Cross-talk between CD14 and complement receptor 3 promotes phagocytosis of mycobacteria: regulation by phosphatidylinositol 3-kinase and cytohesin-1.

The glycosylphosphatidyl anchored molecule CD14 to the monocyte membrane plays a prominent role in innate immunity, and the paradigms for CD14 selective signaling are beginning to be elucidated. In this study, transfected human monocytic cell line THP-1 and Chinese hamster ovary (CHO) fibroblastic cells were used to examine phagocytosis of Mycobacterium bovis bacillus Calmette-Guerin (BCG). Flow cytometry was combined with molecular and biochemical approaches to demonstrate a dual mechanism for BCG internalization involving either CD14 alone or a CD14-regulated complement receptor (CR)3-dependent pathway. Phagocytosis by CD14-positive THP-1 cells was attenuated by phosphatidylinositol-3 inhibitors LY294002 and wortmannin and experiments using transfected CHO cells showed substantial accumulation of phosphatidylinositol-3,4,5-trisphosphate at the BCG attachment site in CHO cells expressing CD14 and TLR2 suggesting that bacteria bind to CD14 and use TLR2 to initiate a PI3K signaling pathway. Additional experiments using blocking Abs showed that anti-TLR2 Abs inhibit phagocytosis of BCG by THP-1 cells. Furthermore, knockdown of cytohesin-1, a PI3K-regulated adaptor molecule for beta(2) integrin activation, specifically abrogated CD14-regulated CR3 ingestion of BCG consistent with the observation of physical association between CR3 and cytohesin-1 in cells stimulated with mycobacterial surface components. These findings reveal that mycobacteria promote their uptake through a process of "inside-out" signaling involving CD14, TLR2, PI3K, and cytohesin-1. This converts low avidity CR3 into an active receptor leading to increased bacterial internalization.