Immunomodulatory and Antibacterial Effects of Cystatin 9 against Francisella tularensis

Abstract

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.

Cystatins 9 and C as a Novel Immunotherapy Treatment That Protects against Multidrug-Resistant New Delhi Metallo-Beta-Lactamase-1-Producing Klebsiella pneumoniae

“…We were the first investigative team to report that a single i.n. dose of rCST9 (50 pg) attenuated the pathophysiological effects of the deadly human pathogen F. tularensis , resulting in clearance of the pulmonary infection and leading to remarkable improvements in survival rates ( 15 ). The success of these studies led us to examine the protective effects of rCST9 in an experimental mouse model of pneumonia.…”

“…Similarly, we previously reported that rCST9 directly decreased the viability and virulence of the deadly human pathogen F. tularensis Schu 4. Proteomic analysis of F. tularensis exposed to rCST9 showed that the cysteine proteinase inhibitor markedly diminished or eliminated key metabolic proteins in the pathogen, thereby preventing nutritional processing and energy production ( 15 ). rCST9 also interfered with the cell wall synthesis of F. tularensis ( 15 ).…”

“…Proteomic analysis of F. tularensis exposed to rCST9 showed that the cysteine proteinase inhibitor markedly diminished or eliminated key metabolic proteins in the pathogen, thereby preventing nutritional processing and energy production ( 15 ). rCST9 also interfered with the cell wall synthesis of F. tularensis ( 15 ). Therefore, rCST9 weakened the pathogen and rendered it more susceptible to killing by the host.…”

“…Less is known about CST9. We were the first to show the immunomodulatory and antimicrobial effects of recombinant CST9 (rCST9) against pulmonary tularemia ( 15 ). Our findings revealed that a single intranasal (i.n.)…”

“…Our findings revealed that a single intranasal (i.n.) dose of rCST9 during a pulmonary Francisella tularensis infection significantly improved survival outcomes, increased human macrophage killing of F. tularensis , and diminished F. tularensis viability ( 15 ).…”