The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

Schmitt, Deanna; Barnes, Rebecca; Rogerson, Taylor; Haught, Ashley; Mazzella, Leanne; Ford, M.; Gilson, Tricia; Birch, James W.-M.; Sjöstedt, Anders; Reed, Douglas S.; Franks, Jonathan; Stolz, Donna B.; Denvir, James; Fan, Jun; Rekulapally, Swanthana; Primerano, Donald A.; Horzempa, Joseph

doi:10.3389/fcimb.2017.00173

Cited by 14 publications

(32 citation statements)

References 63 publications

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“…Our laboratory has previously elucidated the requirement of the T6SS ii in the invasion of erythrocytes by F. tularensis (21). Expression of mglA is upregulated in Francisella incubated with human erythrocytes and both dotU and iglC (both part of the mglA regulon and genes encoding structural components of the T6SS ii ) are required for invasion of erythrocytes by F. tularensis (21). Since OpiA is an effector of this secretion system, we generated a F. tularensis LVS opiA-null mutant (see Fig.…”

Section: Resultsmentioning

confidence: 99%

“…F. tularensis can infect a broad range of host cells, including lung alveolar cells, kidney cells, hepatocytes, fibroblasts, and erythrocytes (16)(17)(18)(19)(20)(21)(22)(23). Moreover, F. tularensis can survive and replicate within host phagocytes, including macrophages, wherein the bacteria block maturation of the phagosome and subsequently escape this compartment (24)(25)(26)(27).…”

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confidence: 99%

“…The T6SS ii of F. tularensis has been shown to be required for invasion of erythrocytes (21). However, F. tularensis does not appear to replicate within red blood cells (RBCs).…”

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confidence: 99%

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OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

2020

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Francisella tularensis is an intracellular pathogen and the causative agent of tularemia. The F. tularensis type six secretion system (T6SS) is required for a number of host-pathogen interactions, including phagolysosomal escape and invasion of erythrocytes. One known effector of the T6SS, OpiA, has recently been shown to be a phosphatidylinositol-3 kinase. To investigate the role of OpiA in erythrocyte invasion, we constructed an opiA-null mutant in the live vaccine strain, F. tularensis LVS. OpiA was not required for erythrocyte invasion; however, deletion of opiA affected growth of F. tularensis LVS in broth cultures in a medium-dependent manner. We also found that opiA influenced cell size, gentamicin sensitivity, bacterial viability, and the lipid content of F. tularensis. A fluorescently tagged OpiA (OpiA–emerald-green fluorescent protein [EmGFP]) accumulated at the cell poles of F. tularensis, which is consistent with the location of the T6SS. However, OpiA-EmGFP also exhibited a highly dynamic localization, and this fusion protein was detected in erythrocytes and THP-1 cells in vitro, further supporting that OpiA is secreted. Similar to previous reports with F. novicida, our data demonstrated that opiA had a minimal effect on intracellular replication of F. tularensis in host immune cells in vitro. However, THP-1 cells infected with the opiA mutant produced modestly (but significantly) higher levels of the proinflammatory cytokine tumor necrosis factor alpha compared to these host cells infected with wild-type bacteria. We conclude that, in addition to its role in host-pathogen interactions, our results reveal that the function of opiA is central to the biology of F. tularensis bacteria. IMPORTANCE F. tularensis is a pathogenic intracellular pathogen that is of importance for public health and strategic defense. This study characterizes the opiA gene of F. tularensis LVS, an attenuated strain that has been used as a live vaccine but that also shares significant genetic similarity to related Francisella strains that cause human disease. The data presented here provide the first evidence of a T6SS effector protein that affects the physiology of F. tularensis, namely, the growth, cell size, viability, and aminoglycoside resistance of F. tularensis LVS. This study also adds insight into our understanding of OpiA as a determinant of virulence. Finally, the fluorescence fusion constructs presented here will be useful tools for dissecting the role of OpiA in infection.

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OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

2020

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“…However, it is not known if Aa ticks concentrate their blood meal more efficiently than Dv ticks, which could help explain differences in Ft numbers in engorged ticks. Separately, other studies have shown that erythrocyte invasion by Ft may enhance its ability to colonize ticks following a blood meal [63]. Although it is unclear how Ft invades erythrocytes, given that these cells do not phagocytose or endocytose, it is possible that Aa ticks imbibe an increased number of erythrocytes via blood meal concentration, compared to Dv ticks, which also may help explain higher Ft numbers in engorged Aa ticks [63].…”

Section: Discussionmentioning

confidence: 99%

A <em>Francisella tularensis</em> Chitinase Contributes to Bacterial Persistence and Replication in Two Major U.S. Tick Vectors

Huntley¹,

Tully²

2020

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Tick-borne tularemia was first described in 1924. Nearly 100 years later, questions remain about the tick vector(s) that pose(s) the greatest risk for transmitting Francisella tularensis (Ft), the causative agent of tularemia. Additionally, few studies have identified genes/proteins required for Ft to infect, persist, and replicate in ticks. To answer questions about vector competence and Ft transmission by ticks, we infected Dermacentor variabilis (Dv), Amblyomma americanum (Aa), and Haemaphysalis longicornis (Hl; invasive species from Asia) ticks with Ft, finding that although Aa ticks initially become infected with 1-log higher Ft, Ft replicated more robustly in Dv ticks, and did not persist in Hl ticks. In transmission studies, both Dv and Aa ticks efficiently infected naïve mice, causing disease in 57% and 46% of those mice, respectively. We identified a putative Ft chitinase, FTL1793, generated a FTL1793 mutant, and found that FTL1793 was deficient in tick infection, persistence, and replication in ticks. Recombinant FTL1793 exhibited chitinase activity in vitro, suggesting that this chitinase may provide an alternative energy source for Ft in ticks. Taken together, Dv ticks appear to pose a greater risk for harboring and transmitting tularemia and FTL1793 plays a major role in promoting tick infections by Ft.

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“…Markerless deletion iglC was accomplished by sequentially deleting codons 71-140 of both copies of iglC as previously described (Schmitt, Barnes, et al, 2017). The entire open reading frame was not deleted to preserve elements presumed to be required for expression of neighboring genes (Lai, Golovliov, & Sjöstedt, 2004).…”

Section: Methodsmentioning

confidence: 99%

The orange spotted cockroach (Blaptica dubia, Serville 1839) is a permissive experimental host for Francisella tularensis

Eklund

Gilson

Mahdi

et al. 2016

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Francisella tularensis is a zoonotic bacterial pathogen that causes severe disease in a wide range of host animals, including humans. Well-developed murine models of F. tularensis pathogenesis are available, but they do not meet the needs of all investigators. Instead, researchers are increasingly turning to insect host systems to: (1) allow high-throughput that is cost-prohibitive or ethically-questionable in mammals; (2) enable studies of host-pathogen interactions when mammalian facilities are unavailable; and (3) provide valuable information about environmental persistence and transmission. However, the utility of previously-described insect hosts is limited because of temperature restriction, short lifespans, and concerns about the immunological status of insects mass-produced for other purposes. Here, we present a novel host species, the orange spotted (OS) cockroach (Blaptica dubia), that overcomes these limitations and is readily infected by F. tularensis. Intrahemocoel inoculation was accomplished using standard laboratory equipment and lethality was directly proportional to the number of bacteria injected. Disease progression differed in insects housed at low and high temperatures, a pattern indicative of a switch between virulence and transmission phenotypes. As in mammalian hosts, F. tularensis mutants lacking key virulence components were attenuated in OS cockroaches. Finally, antibiotics were delivered to infected OS cockroaches by systemic injection and controlled feeding; in the latter case, protection correlated with oral bioavailability in mammals. Collectively, these results demonstrate that this new host system should facilitate discovery of factors that control F. tularensis virulence, immune evasion, and transmission while also providing a platform for early stage drug discovery and development.

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The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

Cited by 14 publications

References 63 publications

OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

OpiA, a Type Six Secretion System Substrate, Localizes to the Cell Pole and Plays a Role in Bacterial Growth and Viability inFrancisella tularensisLVS

A <em>Francisella tularensis</em> Chitinase Contributes to Bacterial Persistence and Replication in Two Major U.S. Tick Vectors

The orange spotted cockroach (Blaptica dubia, Serville 1839) is a permissive experimental host for Francisella tularensis

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