Mycobacterium tuberculosis (Mtb), one of the major pathogens of mankind, kills approximately 2 million people each year. Mtb induces inflammation at the site of infection, leading to leakage of serum proteins, which in turn, are likely to come in contact with the pathogen, thus modulate the pathogenesis of tuberculosis. We studied some of these proteins such as surfactant protein D (SP-D), complement protein C1q and fibronectin, which are either produced locally or they leak-out from serum during inflammation, for their interaction with M.smegmatis and BCG. These non-pathogenic mycobacteria were used as model for Mtb. In this study, the recombinant form of truncated human surfactant protein D (rhSP-D) and three globular heads of human C1q (ghA, ghB, and ghC) were expressed in E.coli. The interaction of each of these proteins with mycobacteria and human monocytic cell line THP-1, was examined via ELISA. We demonstrated that rhSP-D, C1q, three globular heads of C1q and fibronectin bind with both mycobacteria and THP-1 cells. Moreover, using rhSP-D and globular heads of C1q, the binding of SP-D and C1q was localised to C-terminal globular regions. The direct effect for each of these proteins on mycobacterial growth, their effect on the uptake and intracellular fate of mycobacteria inside THP-1 cells were also investigated. Direct interaction of rhSP-D and C1q inhibited mycobacterial growth, whereas fibronectin interaction with the mycobacteria increased their growth. RhSP-D inhibited the uptake and growth of mycobacteria inside THP-1 cells, whereas C1q and each individual globular heads of C1q enhanced the uptake of mycobacteria by THP-1 cells. However, C1q protein inhibited BCG growth but enhanced M.smegmatis growth inside these cells and the later activity was localised to ghA. Fibronectin increased the uptake and growth of mycobacteria inside THP-1 cells. Examining the gene expression of inducible nitric oxide synthase, pro-inflammatory and anti-inflammatory cytokines produced by THP-1 cells infected with the proteins treated and untreated mycobacteria, along with cytokine neutralization experiments, suggest that the nitric oxide components and cytokines could be responsible for mycobacterial growth control inside THP-1 cells. These novel and interesting functions of SP-D, C1q, and fibronectin on mycobacteria provide an insight into the modulatory function of these proteins on Mtb infection, and, therefore, in the pathogenesis of tuberculosis.

The Type III secretion system (T3SS) is an important virulence mechanism that allows pathogenic bacteria to translocate virulence effectors directly into the cytoplasm of eukaryotic host cells to manipulate the host cells in favor of the pathogen. Enteropathogenic Yersinia pseudotuberculosis use a T3SS to translocate effectors, Yops, that prevent phagocytosis by immune cells, and is largely dependent on it to establish and sustain an infection in the lymphoid tissues of a mammalian host. Translocation into a host cell requires specific translocator proteins, and is tightly controlled from both the bacterial and host cell cytoplasm. We aimed to investigate two of the regulatory elements, YopN and LcrV, to gain more insight into the translocation mechanism. Two separate regulatory complexes regulate expression and secretion of Yops, however, the processes are linked so that expression is induced when secretion is activated. A complex, including YopD, prevents expression of Yops, while YopN-TyeA and LcrG block secretion. LcrV is required to relieve the secretion block, by sequestering LcrG. We verified that LcrG binds to the C-terminal part of LcrV, which is consistent with what has been shown in Y. pestis. In addition to their regulatory roles, both LcrV and YopD are translocators and are assumed to interact at the bacterial surface, where LcrV promotes insertion of YopB and YopD into the host cell membrane. However, here we show that purified YopD failed to interact with LcrV, instead YopD solely interacted with a complex of LcrV-LcrG. This indicates that LcrV and YopD interact in the bacterial cytosol, which may be important for regulation of Yop expression and secretion. The established role of YopN is to block secretion prior to host cell contact. We found that deleting the central region (amino acids 76-181) had no effect on the regulatory role of YopN in expression and secretion of Yops. Interestingly, we found that, even though the YopN∆76-181 mutant secreted the translocators with similar kinetics as the wild type strain, translocation of the effector YopH, into HeLa cells, was significantly reduced. Consequently, the YopN∆76-181 mutant was unable to block phagocytosis, almost to the same level as the ∆lcrV mutant which is completely unable to translocate YopH. Our results indicate that YopN is involved in the translocation step in addition to its role in regulating secretion. Further, we show that the amino terminal of LcrV, in the context of translocation, is involved in the early intracellular targeting of YopH in order to block phagocytosis efficiently and sustain an in vivo infection. LcrV mutants that failed to efficiently target YopH intracellularly were severely attenuated also for in vivo virulence. All together, we show that LcrV and YopN are involved in more steps in the regulation of translocation, than what was known before. Our studies also highlight that early translocation is essential for Yersinia to block phagocytosis, which in the end is essential for in vivo virulence.