The transcriptomic response of Streptococcus pneumoniae following exposure to cigarette smoke extract

Abstract

Exposure to cigarette smoke is a risk factor for respiratory diseases. Although most research has focused on its effects on the host, cigarette smoke can also directly affect respiratory pathogens, in some cases enhancing virulence. Streptococcus pneumoniae (the pneumococcus) is a leading cause of community-acquired pneumonia worldwide, however data on the effects of cigarette smoke on the pneumococcus are sparse. Using RNA-seq, we show that pneumococci exposed to cigarette smoke extract in a concentrated acute exposure in vitro model initiate a 'survival' transcriptional response including the upregulation of detoxification enzymes, efflux pumps and osmoregulator transporters, as well as the downregulation of fatty acid and D-alanyl lipoteichoic acid biosynthesis genes. Except for the downregulation of the pneumolysin gene, there were no changes in the expression of major virulence factors following exposure to cigarette smoke. Compared to unexposed pneumococci, smoke-exposed pneumococci did not exhibit any changes in viability, adherence, hydrophobicity or cell lysis susceptibility. In this study, we demonstrate that pneumococci adapt to acute noxious cigarette smoke exposure by inducing a gene expression signature that allows the bacteria to resist its harmful effects.

Figure 1

Effects of cigarette smoke exposure on pneumococcal phenotypes. Log-phase cultures of EF3030 were incubated in CSE or THY media for 45 min. Following incubation, cultures were assayed for (A) viability, (B) adherence to A549 lung epithelial cells, (C) hydrophobicity and (D) sensitivity to treatment with 0.005% Triton X-100. Data in panel A are from three independent experiments, presented as the median with the interquartile range, and analyzed by Mann-Whitney test. All other data (panels B–F) are from ≥3 independent experiments, presented as the mean with standard deviation and analyzed by unpaired t test.

Figure 2

qRT-PCR analysis in strains EF3030 and PMP1287. Genes tested were a selection of 13 genes that were upregulated (A) and downregulated (B) using RNA-seq following exposure to CSE. Selected genes were; SPCG_RS00380 (glyoxalase), SPCG_RS09490 (czcD), SPCG_RS09520 (MarR family transcriptional regulator), SPCG_RS00385 (Metal sensitive transcriptional repressor), SPCG_RS10300 (two-component system transcriptional response regulator 11), SPCG_RS10305 (two-component system sensor histidine kinase 11), SPCG_RS10625 (comGC), SPCG_RS09065 (MarR family transcriptional regulator), SPCG_RS02210 (accA), SPCG_RS09950 (ply), SPCG_RS11330 (dltB), SPCG_RS02155 (fabT) and SPCG_RS09945 (YebC/PmpR family transcriptional regulator). Pneumococci were exposed to CSE or control media for 45 min prior to RNA extraction. Data were normalized to the gyrA gene using the 2−ΔΔCt method. Data are presented as the mean fold change ± standard deviation in cultures incubated in CSE relative to those incubated in THY control media (n = 3 independent experiments). Dotted line represents the biological significance threshold (log₂ = 1 or −1). Confidence intervals are provided in Table S2.