Comparative Phosphoproteomics Reveals Components of Host Cell Invasion and Post-transcriptional Regulation During Francisella Infection

Abstract

Francisella tularensis is a facultative intracellular bacterium that causes the deadly disease tularemia. Most evidence suggests that Francisella is not well recognized by the innate immune system that normally leads to cytokine expression and cell death. In previous work, we identified new bacterial factors that were hyper-cytotoxic to macrophages. Four of the identified hyper-cytotoxic strains (lpcC, manB, manC, and kdtA) had an impaired lipopolysaccharide (LPS) synthesis and produced an exposed lipid A lacking the O-antigen. These mutants were not only hyper-cytotoxic but also were phagocytosed at much higher rates compared with the wild type parent strain. To elucidate the cellular signaling underlying this enhanced phagocytosis and cell death, we performed a large-scale comparative phosphoproteomic analysis of cells infected with wild-type and delta-lpcC F. novicida. Our data suggest that not only actin but also intermediate filaments Francisella tularensis is the Gram-negative facultative intracellular bacterium that causes the disease tularemia in humans and diverse animals. This bacterium is among the most virulent human pathogens known, with inhalation or inoculation of as few as 10 bacteria being sufficient to cause a fatal infection. Its low infectious dose and relative ease of airborne transmission led to the development of F. tularensis as a biological weapon (1). Because of potential threats to national security and public health, the U.S. Centers for Disease Control and Prevention (CDC) classifies F. tularensis as a Category A bioterrorism agent, and the Departments of Health and Human Services (HHS) and Agriculture (USDA) list this pathogen as a Tier 1 select agent. (http://www.bt.cdc.gov/agent/ agentlist-category.asp; http://www.selectagents.gov/select% 20agents%20and%20toxins%20list.html).Francisella tularensis includes three subspecies: tularensis (abbreviated as F. tularensis), holarctica (abbreviated as F. holarctica), and mediasiatica (abbreviated as F. mediasiatica) (2). Of these, F. tularensis subsp. tularensis and subsp. holarctica are capable of causing disease in humans and must be manipulated in a Biosafety Level 3 environment. Francisella novicida (abbreviated as F. novicida) is a closely related species to F. tularensis and shares high genetic similarity to other F. tularensis subspecies (ϳ98% DNA sequence identity) (3, 4). Although some differences have been observed so far between F. novicida and F. tularensis, mainly in their lipopolysaccharide (LPS) 1 O-antigen and the ability to activate the inflammasome, most of the mutations in orthologous genes generate similar phenotypes (5-7). Because of the avirulence in humans for the U112 isolate (8)

Virulent <b><i>Francisella tularensis</i></b> Destabilize Host mRNA to Rapidly Suppress Inflammation

“…Rather, there is precedent in the literature for increasing mRNA stability by bacteria and/or bacterial components as a mechanism to sustain inflammatory responses. For example, avirulent F. novicida elicits a strong pro-inflammatory response following infection of host cells and it has been proposed that F. novicida inactivates TTP as a mechanism to enhance pro-inflammatory responses [34–36]. Specifically, a F. novicida ΔlpcC mutant provokes an even greater inflammatory response compared to wild type F. novicida .…”

Global Reprogramming of Host Kinase Signaling in Response to Fungal Infection

“…(Schmutz et al, 2013), Francisella at 2 h.p.i. (Nakayasu et al, 2013), Salmonella at 8 h.p.i. (Rogers et al, 2011), or HIV-1 at 1 m.p.i.…”

Multiple mechanisms drive phage infection efficiency in nearly identical hosts

“…For proteomics quantitation by iTRAQ methods (for phi38:1 on C. baltica NN016038 and #18), the label-free quantitation approach was followed except higher energy collisional dissociation was used for peptide fragmentation [57,58]. All peptide database searches used the C. baltica and phage genomes described above.…”