Analysis of the effects of cigarette smoke on staphylococcal virulence phenotypes

Abstract

Cigarette smoking is the leading preventable cause of death, disease, and disability worldwide. It is well established that cigarette smoke provokes inflammatory activation and impairs antimicrobial functions of human immune cells. Here we explore whether cigarette smoke likewise affects the virulence properties of an important human pathogen, Staphylococcus aureus, and in particular methicillin-resistant S. aureus (MRSA), one of the leading causes of invasive bacterial infections. MRSA colonizes the nasopharynx and is thus exposed to inhalants, including cigarette smoke. MRSA exposed to cigarette smoke extract (CSE-MRSA) was more resistant to macrophage killing (4-fold higher survival; P < 0.0001). CSE-MRSA demonstrated reduced susceptibility to cell lysis (1.78-fold; P = 0.032) and antimicrobial peptide (AMP) (LL-37) killing (MIC, 8 μM versus 4 μM). CSE modified the surface charge of MRSA in a dose-dependent fashion, impairing the binding of particles with charge similar to that of AMPs by 90% (P < 0.0001). These changes persisted for 24 h postexposure, suggesting heritable modifications. CSE exposure increased hydrophobicity by 55% (P < 0.0001), which complemented findings of increased MRSA adherence and invasion of epithelial cells. CSE induced upregulation of mprF, consistent with increased MRSA AMP resistance. S. aureus without mprF had no change in surface charge upon exposure to CSE. In vivo, CSE-MRSA pneumonia induced higher mouse mortality (40% versus 10%) and increased bacterial burden at 8 and 20 h postinfection compared to control MRSA-infected mice (P < 0.01). We conclude that cigarette smoke-induced immune resistance phenotypes in MRSA may be an additional factor contributing to susceptibility to infectious disease in cigarette smokers.

FIG 1

Cigarette smoke exposure reduces MRSA susceptibility to macrophage killing and lysis while suppressing bacterial growth. (A) Starting with an MOI of 1 × 10⁵, control MRSA was killed by alveolar macrophages over 100 min, while MRSA exposed to 75% CSE resisted killing and overgrew. By 220 min, CFU of control MRSA were 4-fold lower than those of CSE-MRSA. (B) CSE exposure did not change the rate of MRSA-GFP phagocytosis by macrophages. (C) The increased numbers of MRSA during killing assays was not due to increased growth, as CSE decreased the rate of MRSA growth in a dose-dependent manner. (D) CSE exposure tended to increase MRSA resistance to killing by H₂O₂ (oxygen radicals). (E) CSE induced resistance to MRSA cell lysis in the presence of detergent. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 2

Cigarette smoke exposure increases resistance of MRSA to human AMP LL-37. (A) CSE exposure increased the MIC of MRSA to LL-37 from 4 to 8 μM and increased the concentration needed to inhibit metabolic activity from 8 to 16 μM. (B) Growth of control MRSA and MRSA after exposure to 75% CSE was identical during the AMP growth kinetics assays, which were run without CSE present. (C, D, and F) Prior exposure to 75% CSE allowed MRSA to escape killing by LL-37 at 16 μM (C), 8 μM (D), and 2 μM (F). (E and G) These effects were CSE dose dependent, with exposure to 50% through 90% CSE inducing resistance to AMP killing at both 8 μM (E) and 2 μM (G). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Cigarette smoke exposure induces MRSA surface changes, including a less negative surface charge and increased hydrophobicity, leading to increased adherence and invasion of epithelial cells. (A) CSE-MRSA binds less of cationic PLL-FITC, consistent with alteration in surface charge (less negative = more positive surface charge) in response to CSE exposure, in a dose-dependent manner. (B) Background fluorescence (no PLL added) was equal among groups. CSE precipitate alone had no impact on surface charge, with equivalent surface binding of 2 μM PLL as control MRSA. However, 24-h-old CSE induced a 2-fold shift in surface charge, but not the 12-fold shift induced by freshly made CSE. (C) Nicotine alone induced shifts in MRSA surface charge leading to less PLL-FITC binding (5-fold by 3 mg and 11-fold by 6 mg), consistent with nicotine contributing to CSE-induced surface charge changes. (D) Surface charge changes induced by CSE exposure persisted after passage into THB two times, for 24 h after exposure, and repetitive daily exposures for 3 and 4 days further enhanced surface charge changes. (E) S. aureus strain SA113 had similar changes in PLL-FITC binding upon exposure to CSE. (F) Exposure to CSE induced increasing hydrophobicity in a dose-dependent manner, as evidenced by fewer bacteria recovered from the aqueous phase as the CSE concentration was increased. (G and H) CSE exposure increased adherence of MRSA to epithelial cells (G) and increased MRSA invasion of and persistence within epithelial cells (H). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 4

MRSA exposed to CSE has increased virulence in a mouse model of pneumonia. (A) Lungs harvested at 8 and 20 h postinfection had higher numbers of CSE-MRSA (**, P < 0.01). (B) In a mortality model of pneumonia, 40% of mice infected with CSE-MRSA died, whereas 10% of controls survived (P = 0.021; n = 20 mice per group). (C) Histology of lungs from mice with MRSA pneumonia demonstrated more bacteria and inflammation in CSE-MRSA-infected mice at both the 8- and 20-h time points (n = 5 per group at each time point) (inflammation scores shown in white).