Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

Abstract

bCertain intracellular bacteria use the host cell cytosol as the replicative niche. Although it has been hypothesized that the successful exploitation of this compartment requires a unique metabolic adaptation, supportive evidence is lacking. For Francisella tularensis, many genes of the Francisella pathogenicity island (FPI) are essential for intracellular growth, and therefore, FPI mutants are useful tools for understanding the prerequisites of intracytosolic replication. We compared the growth of bacteria taken up by phagocytic or nonphagocytic cells with that of bacteria microinjected directly into the host cytosol, using the live vaccine strain (LVS) of F. tularensis; five selected FPI mutants thereof, i.e., ⌬iglA, ⌬iglC¸⌬iglG, ⌬iglI, and ⌬pdpE strains; and Listeria monocytogenes. After uptake in bone marrow-derived macrophages (BMDM), ASC ؊/؊ BMDM, MyD88 ؊/؊ BMDM, J774 cells, or HeLa cells, LVS, ⌬pdpE and ⌬iglG mutants, and L. monocytogenes replicated efficiently in all five cell types, whereas the ⌬iglA and ⌬iglC mutants showed no replication. After microinjection, all 7 strains showed effective replication in J774 macrophages, ASC ؊/؊ BMDM, and HeLa cells. In contrast to the rapid replication in other cell types, L. monocytogenes showed no replication in MyD88 ؊/؊ BMDM and LVS showed no replication in either BMDM or MyD88 ؊/؊ BMDM after microinjection. Our data suggest that the mechanisms of bacterial uptake as well as the permissiveness of the cytosolic compartment per se are important factors for the intracytosolic replication. Notably, none of the investigated FPI proteins was found to be essential for intracytosolic replication after microinjection. Bacteria and other microbes have developed an ability to invade host cells and use them as a principal habitat for replication. These so-called intracellular pathogens are able to trigger their uptake by mammalian cells, by phagocytosis when the host cells are professional phagocytes, e.g., monocytes or macrophages, or by triggered phagocytosis in the case of nonprofessional phagocytic host cells, such as epithelial or endothelial cells, hepatocytes, and fibroblasts (1, 2). After internalization, virulence factors produced by the intracellular pathogen modulate the intracellular environment to facilitate microbial survival (3, 4). For protection against intracellularly located microorganisms, the immune system is dependent on pattern recognition receptors (PRR) that identify conserved microbial components (5). The best-characterized family of PRR is the one of Toll-like receptors (TLR), a group of integral membrane proteins that recognize microbial components, such as lipopolysaccharide, bacterial lipoprotein, and CpG DNA (6, 7). Triggering of TLR leads to rapid initiation of an antimicrobial proinflammatory response (8, 9). These innate defense mechanisms are normally sufficient to mediate the eradication of phagocytosed extracellular pathogens but not to control those capable of intracellular replication.Many intracellular bacteria, e.g., Mycobacterium,...

Secreted Effectors Encoded within and outside of the Francisella Pathogenicity Island Promote Intramacrophage Growth

“…A wealth of data indicate that T6SS ii is required for endosomal escape of Francisella ; however, there are conflicting data pertaining to its requirement for establishing a replicative cytoplasmic niche (Meyer et al, 2015; Wu et al, 2015). We found that a strain lacking each of the five effectors identified in our secretome analysis retains core apparatus function, but displays phagosomal escape and intracellular growth defects equal to or greater than strains containing an inactive T6SS ii apparatus.…”

Critical Role of a Sheath Phosphorylation Site On the Assembly and Function of an Atypical Type VI Secretion System

“…Of note, the FPI is duplicated in three subspecies of F. tularensis (subsps holarctica, mediasiatica, and tularensis), but is present in a single copy in F. novicida and F. philomiragia. The FPI encodes a Type VI secretion system (T6SS) that is essential to promote bacterial phagosomal escape and access to the cytosolic replication niche (19)(20)(21). Whereas the transcriptional regulation of the FPI locus has been deeply characterized (22), the molecular mechanisms triggering T6SS assembly and contraction remain largely unknown.…”

A post-translational modification of the sheath modulatesFrancisellatype VI secretion system assembly and function

“…Nevertheless, some T6SS elements are likely to be required during the cytosolic stage of the infectious cycle. Indeed, most FPI genes are induced during late intra-macrophage growth (Wehrly, Chong et al, 2009) and IglC has been shown to be required for intracellular growth of F. novicida that are microinjected directly into the cytosol of HeLa cells (Meyer et al, 2015). IglC secretion in F. novicida depends on the T6SS core components IglA and IglB (Ludu, de Bruin et al, 2008).…”

“…It is also possible that a single kinase mutant might have no or very limited effect on phagosomal disruption since sheath polymerization would occur and be sufficient to promote phagosomal membrane disruption. Indeed, it has been shown (Meyer et al, 2015) that only a minor fraction of functional T6SS is sufficient to promote phagosomal escape and, once in the cytosol, FPI mutants can multiply like wild-type bacteria.…”

“…The FPI encodes a Type VI secretion sytem (T6SS) that is essential to promote bacterial phagosomal escape and access to the cytosolic replication niche (Lauriano, Barker et al, 2004, Meyer, Broms et al, 2015, Nano, Zhang et al, 2004). Whereas the transcriptional regulation of the FPI locus has been deeply characterized (Ramsey, Osborne et al, 2015), the molecular mechanisms triggering T6SS assembly and contraction remain largely unknown.…”