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Abstract Tularemia is a zoonosis caused by CDC-declared Tier 1 threat agent Francisella tularensis. F. tularensis subsp. novicida (F. novicida) is virulent in mice but non-pathogenic in immunocompetent humans and serves as a potential surrogate organism. In a recent study, we established a silkworm (Bombyx mori) model of infection for F. novicida. Francisella secretes its virulence factors through various mechanisms that modify the intracellular environment to ensure its replication and survival. To identify new pathogenic factors, we focused on the type I secretory system (T1SS) of Francisella. In silico analysis revealed a RtxA (Repeats-in-toxin) like protein in the Francisella genome. The characteristics of RtxA like protein were investigated using mutant analysis. Firstly, the role of rtxA in silkworms was investigated by infecting them with F. novicida strains into the hemocoel. The rtxA mutant failed to kill the silkworms, whereas F. novicida wild-type (WT) strain killed silkworms within 3–7 days post infection. The arrested growth of the mutant strain in silkworms was observed using a whole-body CFU count assay. We also investigated the growth characteristics of the rtxA mutant in hemocytes, one of the primary multiplication sites of Francisella within silkworms. Interrupted growth of the rtxA mutant with significantly reduced cytotoxicity was observed in hemocytes via confocal microscopy. Next, we analyzed the effect of rtxA in human monocyte cell line THP-1. The mutant strain showed significantly decreased growth and reduced cytotoxicity compared with its parental strain in THP-1 cells. This study newly identified RtxA like protein of F. novicida as an important lethal pathogenic factor in silkworm and mammalian cells. Highlights • There is a RtxA like protein in Francisella novicida. • The deletion mutant of RtxA like protein failed to kill silkworm. • RtxA like protein contributes to intracellular growth in THP-1 cells. [ABSTRACT FROM AUTHOR]

Understanding the virulence and pathogenesis of human pathogens using insect models is an increasingly popular method. Francisella novicida , which is virulent in mice but non-pathogenic to immunocompetent humans, is widely used as an ideal candidate for Francisella research. In this study, we developed a silkworm ( Bombyx mori ) infection model for F. novicida by inoculating the hemocoels of silkworms with F. novicida . We found that silkworms died within 3–7 days of F. novicida infection. However, the deletion mutant of DotU, the core part of type VI secretion systems, failed to kill silkworm. In whole silkworm bodies, the bacterial load of the DotU deletion mutant was significantly less than that of the wild-type strain. Approximately 10-fold increase in bacterial load was recorded in hemolymph and subcutaneous tissues compared with that in the silk gland, Malpighian tubule, and reproductive organs. The CFU count of the DotU deletion mutant in all organs was similar results to the whole body CFU count. Confocal microscopy further confirmed the arrested growth of the mutant strain within hemocytes. The intracellular growth of F. novicida strains was also analyzed using the silkworm ovary-derived cell line BmN4. In BmN4, both CFU count assay and confocal microscopy revealed extensive growth of the wild-type strain compared with that of the mutant strain. Francisella DotU has already been proven as a virulence factor in mammals, and it was also found to be an essential virulence factor in our silkworm infection model. Therefore, this silkworm infection model is suitable for identifying new virulence factors of Francisella . [ABSTRACT FROM AUTHOR]

Francisella novicida is a gram-negative pathogen commonly used to study infections by the potential bioterrorism agent, Francisella tularensis . The Francisella lipid A structure has been well characterized and showed to affect the pathogenesis of F. novicida . Previous work characterized two lipid A acyltransferases, LpxD1 and LpxD2, and constructed the lpxD1-null and lpxD2-null mutants. Mutational analysis showed the lpxD1-null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. However, details as how the virulence has been changed have remained elusive. This study aims to analyze effects of lipid A acyltransferases on the pathogenesis of F. novicida . MS and MS n were conducted to confirm the lipid A structures of lpxD1-null and lpxD2-null mutants. The stress tolerance, Toll-like receptor 4 (TLR4) stimulation level, intracellular survival and replication ability and cytotoxicity of lpxD1-null and lpxD2-null mutants were analyzed. The results suggested the lpxD1-null mutant with shorter acyl chains in lipid A is more sensitive to various environmental stresses than F. novicida and lpxD2-null mutant. In addition, the lpxD1-null mutant fails to survive and replicate in cells and shows lower cytotoxicity to infected cells. This study provides insights into the pathogenesis of F. novicida . [ABSTRACT FROM AUTHOR]