B. pseudomallei epidemic area is reported to be expanding in tropical and subtropical countries worldwide [22], including the South China Sea and Taiwan Strait [23, 24], and has been listed as a Tier-1 (top tier) select agent [2]. The intracellular lifestyle of B. pseudomallei in host cells has been reported to impact the severity of melioidosis, ranging from acute fatal sepsis to chronic infection with or without clinical symptoms [21, 25, 26]. However, the information about how B. pseudomallei impact on host cell responses is still limited during the whole intracellular infection processare. In this study, B. pseudomallei infection of a human lung epithelial A549 cell model was used for the global transcriptomic responses at early infected stage and late infected stage in contrast to control cells. We found the differential expression genes mainly involved in inflammation and stress response.
The A549 cell line is known to be a highly susceptible to infection of B. pseudomallei [3, 12, 27], and researches about many other intracellular bacteria infection also used A549 cell model. In the current study, the transcriptomic kinetics following lastingness of A549 infected with B. pseudomallei was conducted by GeneChip Human Gene 2.0 ST Arrays from Affymetrix, which represents approximately 30,654 transcribed genes. Microarray technology for transcriptome yields a large amount of data, and it is important to validate differential expression by independent methods, which conformed to the results of microarray assay. Real-time quantitative PCR analysis of random ten DEGs, including IL-6 and TNF-α (Fig. 2C), were therefore conducted, although there were fold differences between the RT-qPCR and transcriptome data, the result indicated that the expression profiles of differentially expressed genes in A549 were notably changed with the infection of B. pseudomallei.
In general, innate immune mechanisms are critical in determining the outcome of infections caused by bacterial pathogens. Recent studies have reported that B. pseudomallei increased the production of inflammatory cytokines such as TNF-α, IL-1β and IL-6 in vivo in serum samples of melioidosis patients [28]. Proinflammatory cytokine mRNA of TNF-α, IL-1β and IL-6 increased in the liver of mice following infected with B. pseudomallei [29]. In agreement with previous studies, our results showed that B. pseudomallei elevated IL-1β level and increased the mRNA expression of TNFAIP3 and IL-6 in human lung epithelial cell A549. Several lines of evidence suggest that one major role of eIFα/ATF4 pathway is to mediate the induction of a gene expression program referred to as the integrate stress response (ISR) [30, 31], involved in amino acid deficiency, endoplasmic reticulum stress (ERS), oxidative stress and drug resistance [32–34]. Our team's previous research indicated that B. pseudomallei could enhance NF-κB pathway in RAW264.7 macrophage cell through down-regulating TRAF3, a well-known negative regulator of the NF-κB, via increasing miR-3473 [35]. We further validated the transcriptional activator in inflammation, including p-eIF2α, ATF4 and NF-κB2. The data yield that B. pseudomallei exposure could active the eIF2α/ATF4 pathway and NF-κB2. B. pseudomallei may adjust host inflammation and stress response to cope with the intracellular encountered stress.
Previous studies have reported B. pseudomallei can in fact induce cell fusion leading to MGCs formation in both phagocytic and nonphagocytic cell lines [12, 36]. Cell fusion and the formation of MGCs have long been regarded as a possible mechanism for B. pseudomallei to cell-to-cell spreading [21]. In the present study, we found B. pseudomallei could induce A549 cells fusion, resulting in the formation of MGCs at the late infected stage, and B. pseudomallei were massively replicated in MGCs, which was consistent with previous studies. Additionally, The formation of MGCs was also found to exist in many other intracellular infectious agents such as human immunodeficiency virus (HIV), cytomegalovirus (CMV) and herpes simplex virus (HSV) [37–39]. It is reported that persistent existence of mycobacterial species leads to the formation of MGCs which play important roles in many physiological and pathological processes [40]. Meanwhile, B. pseudomallei could form filamentous actin tail whether in the early or late stage of infection, as seen in the Fig. 1A. Studies have shown that actin-based motility is an essential prerequisite for the production of MGCs and the spread of B. pseudomallei from cell-to-cell [21]. And recent evidences indicate that actin-based motility of B. pseudomallei is dependent upon the autosecreted protein, BimA [41]. While it still remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote B. pseudomallei persistence. In our study, the formation of MGCs induced by B. pseudomallei may be a characteristic feature at chronic inflammatory sites in A549 cells to escape from intracellular immune defense mechanisms or help B. pseudomallei accessed to nutrients to achieve replication.