The p60 and NamA autolysins fromListeria monocytogenescontribute to host colonization and induction of protective memory

Abstract

Inducing long-term protective memory CD8+ T cells is a desirable goal for vaccines against intracellular pathogens. However, the mechanisms of differentiation of CD8+ T cells into long-lived memory cells capable of mediating protection of immunized hosts remain incompletely understood. We have developed an experimental system using mice immunized with WT or mutants of the intracellular bacterium Listeria monocytogenes (Lm) that either do or do not develop protective memory CD8+ T cells. We previously reported that mice immunized with Lm lacking functional SecA2, an auxiliary secretion system of gram-positive bacteria, did not differentiate functional memory CD8+ T cells that protected against a challenge infection with WT Lm. Herein we hypothesized that the p60 and NamA autolysins of Lm, which are major substrates of the SecA2 pathway, account for this phenotype. We generated Lm genetically deficient for genes encoding for the p60 and NamA proteins, ΔiapΔmurA Lm, and further characterized this mutant. Δp60ΔNamA Lm exhibited a strong filamentous phenotype, inefficiently colonized host tissues, and grew mostly outside cells. When Δp60ΔNamA Lm was made single unit (SU), cell invasion was restored to WT levels during vaccination, yet induced memory T cells still did not protect immunized hosts against recall infection. Recruitment of blood phagocytes and antigen-presenting cell activation was close to that of mice immunized with ΔActA Lm which develop protective memory. However, key inflammatory factors involved in optimal T cell-programming such as IL-12 and type I IFN (IFN-I) were lacking, suggesting that cytokine signals may largely account for the observed phenotype. Thus altogether, these results establish that p60 and NamA secreted by Lm promote primary host cell-invasion, the inflammatory response and the differentiation of functional memory CD8+ T cells, by preventing Lm filamentation during growth and subsequent triggering of innate sensing mechanisms.

SecA2 Associates with Translating Ribosomes and Contributes to the Secretion of Potent IFN-β Inducing RNAs

“…First, we examined wild type Lm, Lm-∆ secA2 and Lm-∆ secA 2::SecA2 strep for changes in morphology during exponential growth phase using light microscopy. Lm-∆ secA2 form long cell chains due to a septal defect that results from lack of the secretion of p60/Iap and MurA/NamA [ 16 , 17 , 18 ] ( Figure 1 A). The complemented recombinant strain Lm-∆ secA 2::SecA2 strep had reverted to the single cell morphology seen with wild type (Lm) bacteria ( Figure 1 A).…”

“…L. monocytogenes secretes 19 proteins in a SecA2-dependent manner [ 4 , 16 ]. The cell wall-secreted hydrolases autolysin p60 (protein of 60 kDa, or Iap (Invasion associated protein) also called CwhA (cell wall hydrolase A)) and the muramidase MurA (also called NamA [N-acetylmuramidase A]), known to be involved in cell division, are well known SecA2 substrates [ 16 , 17 , 18 ]. Listeria lacking SecA2 show reduced secretion of both p60 and MurA in the supernatant of growing bacteria, and exhibit a chain-like cell morphology manifesting in a rough colony type [ 15 , 17 ].…”

“…Listeria lacking SecA2 show reduced secretion of both p60 and MurA in the supernatant of growing bacteria, and exhibit a chain-like cell morphology manifesting in a rough colony type [ 15 , 17 ]. The reduced secretion of SecA2-dependent proteins such as p60 has been implicated in the diminished virulence phenotype [ 16 , 18 , 19 , 20 ]. Additionally, SecA2-inactivation affects significantly the degree of hydrophilicity and Lewis acid-based properties, and thereby affects the cell surface properties of L. monocytogenes [ 21 ].…”

Vaginal microbiome modulates topical antiretroviral drug pharmacokinetics

“…Fractions were dried in a lyophilizer and resuspended to their injection volume. The bioactive fraction was subsequently analyzed by ultra-performance liquid chromatography tandem MS (UPLC-MS/MS) (38). Fractions were separated on a Waters Acquity UPLC system in HILIC mode coupled to a Waters Synapt G2 quadrupole time-of-flight mass spectrometer, which was used to analyze the eluate.…”

The Two Distinct Types of SecA2-Dependent Export Systems

“…Multi-substrate SecA2 systems export more than one substrate, although the number of exported substrates is still small when compared to the general Sec system. The multi-substrate SecA2 systems of Mycobacterium tuberculosis (46, 47), Listeria monocytogenes (48, 49) and likely Bacillus anthracis (50, 51) are required for pathogenesis. In Corynebacterium glutamicum (52) and Clostridium difficile (53) their multi-substrate systems are essential for bacterial viability.…”