Immune lymphocytes halt replication of Francisella tularensis LVS within the cytoplasm of infected macrophages

Abstract

Francisella tularensis is a highly infectious intracellular bacterium that causes tularemia by invading and replicating in mammalian myeloid cells. Francisella primarily invades host macrophages, where it escapes phagosomes within a few hours and replicates in the cytoplasm. Less is known about how Francisella traffics within macrophages or exits into the extracellular environment for further infection. immune t lymphocytes control the replication of Francisella within macrophages in vitro by a variety of mechanisms, but nothing is known about intracellular bacterial trafficking in the face of such immune pressure. Here we used a murine model of infection with a Francisella attenuated live vaccine strain (LVS), which is under study as a human vaccine, to evaluate the hypothesis that immune T cells control intramacrophage bacterial growth by redirecting bacteria into toxic intracellular compartments of infected macrophages. We visualized the interactions of lymphocytes and LVS-infected macrophages using confocal microscopy and characterized LVS intramacrophage trafficking when co-cultured with immune lymphocytes. We focused on the late stages of infection after bacteria escape from phagosomes, through bacterial replication and the death of macrophages. We found that the majority of LVS remained cytosolic in the absence of immune pressure, eventually resulting in macrophage death. In contrast, co-culture of LVS-infected macrophages with LVS-immune lymphocytes halted LVS replication and inhibited the spread of LVS infection between macrophages, but bacteria did not return to vacuoles such as lysosomes or autophagosomes and macrophages did not die. Therefore, immune lymphocytes directly limit intracellular bacterial replication within the cytoplasm of infected macrophages. Francisella tularensis is a gram-negative, facultative intracellular bacterium that replicates in macrophages and causes tularemia in humans 1-3. Francisella can infect people via multiple routes, but respiratory infection leads to the most severe form of the disease and can be fatal if not treated. F. tularensis is found throughout North America and is endemic in Europe, especially in Scandinavia, and in Asia. Tularemia is not a large public health concern in developed countries. However, Francisella was investigated as a bioweapon in the mid-1900s by both the United States and Soviet Union; the bacterium is therefore is currently categorized as a Select Agent in the United States 3. The intracellular lifecycle of Francisella has been visualized in vitro with fixed and live-cell microscopy. Francisella enters macrophages via phagocytosis and escapes from the resulting phagosome within 1 to 4 h, avoiding lysosomal fusion 4-13. Cytosolic Francisella replicates to high numbers over the next day 9. To date, in-depth characterization has focused on the first 24 h after infection of host myeloid cells. One set of reports has described clustering of LVS about 20 h after infection of murine macrophages into "Francisella-containing vacuoles" (FCVs), which ...

Modern Development and Production of a New Live Attenuated Bacterial Vaccine, SCHU S4 ΔclpB, to Prevent Tularemia

“…Clinical scores after vaccination, skin reactogenicity, bacterial genomes ( Supplementary Table S4 ), and levels of cytokines in sera within 4 days after vaccination ( Supplementary Table S5 ) have been monitored, all options that do not depend on lethal infections. Moreover, we have extensively explored the use of an in vitro co-culture assay that measures the ability of lymphocytes from ΔclpB vaccinated mice or rats to control the intramacrophage growth of Francisella bacteria [ 46 , 54 , 71 , 72 , 73 , 74 ]. The degree of bacterial growth control tracks well with the degree of in vivo protection [ 54 , 73 , 75 ], making this assay a potential potency assay.…”

Host Immunity and Francisella tularensis: A Review of Tularemia in Immunocompromised Patients

“…The same Th1 type cytokine production in response to LVS antibody exposure is found in humans [17,40]. Moreover, it has been shown that IFNγ and TNFα contribute to the control of the bacterial growth in macrophage by CD4+ and CD8+ T cells after the initial contact with bacteria [41,42]. Interestingly, the two subsets of T cell respond differently to these factors since CD4+ rely more on IFNγ whereas CD8+ rely almost completely on TNFα [41,43].…”

Cathepsins in Bacteria-Macrophage Interaction: Defenders or Victims of Circumstance?

“…S. aureus is a Gram-positive bacterium causing both communityand hospital-acquired infections with significant morbidity and mortality (Magill et al, 2014a;Magill et al, 2014b). Methicillinresistant S. aureus (MRSA) is responsible for invasive, drugresistant skin and soft tissue infections contributing to the development of diseases, such as endocarditis, osteomyelitis, or bacteremia (Klevens et al, 2007;Brann et al, 2019).…”

“…Interestingly, T cells from vaccinated mice can provide a functional assistance in arresting Francisella attenuated live vaccine strain (LVS) replication and inhibiting the spread of LVS infection between macrophages in in vitro conditions. Coculture experiments of LVS-infected macrophages with naïve or LVS-immune lymphocytes revealed that LVS-immune T cell control of replication and spread of LVS in macrophages is mediated by a direct effect on the viability of bacteria in the cytoplasm, rather than intracellular trafficking of bacteria into lysosomes for degradation in infected macrophages (Bradford and Elkins, 2020).…”