Exploring the impact of paracetamol on the transcriptome of Streptococcus pneumoniae D39

1 Introduction

An opportunistic pathogen of humans, Streptococcus pneumoniae, inhabits the mucosa of nasal portions and causes sinusitis, otitis media, sepsis, meningitis and pneumonia which results about a million people deaths every year worldwide (O'Brien et al., 2009). Multi-resistant pneumococcal strains’ emergence and dissemination has been on the rise since mainstream drugs usage worldwide (Kim et al., 2016). Generation of resistant pneumococcal clones results through adaptation to drug pressures enforced although they reside within the human upper respiratory tract (Kim et al., 2016). Most pneumococcal antimicrobial resistances have basic causal factors that have been uncovered (Kim et al., 2016). The escalating rates of resistance to antibiotics to all currently available treatments, along with an almost empty pharmacological pathways for new drugs, has caused a panic in drug discovery efforts around the world (Brockhurst et al., 2019). Though different drugs have shown potency against most pneumococcal infections, the existing options are limited against some pneumococcal isolates (Kim et al., 2016).

Several factors are deemed liable for the development of resistance among intrusive pneumococcal disease cases including modern drug usage (foremost risk factor) (Levine et al., 1999), age (predominantly children below 5 years of age) and pediatric serotypes (serotypes found commonly in children), hospitalization, attending day care, female gender, living in an urban area, HIV infection and immunosuppression (Levine et al., 1999). At present, >40 % of pneumococcal isolates lack significant conjugate vaccine coverage and are penicillin resistant in several countries (Torné et al., 2014). Paracetamol is deemed as the first-line remedy for an acute sore throat. Nonetheless, in primary care, antibiotics are still normally prescribed as first-line treatment for sore throat. Therefore, we believe that it would be very interesting to give a try to a less-problematic and routinely used medication paracetamol (acetaminophen/APAP) and investigate the response of pneumococcus to paracetamol at molecular level. Moreover, paracetamol has been shown to enhance biofilm formation in human pathogen Staphylococcus aureus. This research elucidates the impact of paracetamol on the whole transcriptome of pneumococcus and highlights the putative targets of paracetamol in pneumococcus by differential network analysis and pathway enrichment analysis.

2 Methods

3 Results

3.2

3.2 PPI network analysis of the differentially expressed genes

PPI networks have been utilized to study health and disease of a certain body and can be very handy for comparison of systems across diverse conditions. The STRING database was used to build PPI networks in order to better understand the connections of differentially expressed genes. As shown in Fig. 1A, the hub genes with node degree greater than or equal to 10 fatty acid biosynthesis and metabolism genes. These genes include spd-0378, acpP, fabH, fabK, fabD, fabF, fabG, accB, accC, fabZ, accD and accA. Another network that was prominent in our analysis was the one consisting of genes involved in the biosynthesis of antibiotics. This network includes fabD, fabG, accA, accB, accC, accD, spd-1834 (adhE), dapA, asd, ilvA, ilvB, ilvC, ilvE and ilvN. Both fatty acid biosynthesis and metabolism genes and antibiotics biosynthesis genes’ networks have been shown in separate figures (Fig. 1B and 1C, respectively).

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Fig. 1

Protein-protein interaction (PPI) differential network analysis of the pneumococcal transcriptome in response to 5 mM paracetamol in the medium (A). PPI differential network analysis of the fatty acid genes (B) and the antibiotic biosynthesis genes (C).

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4 Discussion

Growing drug resistance is a major challenge in treating pneumococcal throughout the last few decades. Presently, 15–30 % of the pneumococcal strains are categorized as resistant to multiple drugs (Lynch and Zhanel, 2009). Most common drug treatments of pneumococcus involves macrolides, β-lactams, or fluoroquinolones individually or in group (Weiss et al., 2004; Waterer and Rello, 2006). The use of antibiotic combinations broadens the range of bacteria that can be targeted while also increasing efficacy, decreasing the inception and propagation of resistance bacteria. Understanding the response of pneumococci to commonly used drugs may provide novel therapeutic targets and important insights into the adaptive techniques required to interact with the host environment during infection (Leonard and Lalk, 2018). Using paracetamol as a part of combination therapy may be useful. We investigated the relation of S. pneumoniae to paracetamol to identify the potential candidates for drugs against pneumococcus. This research will aid in the knowledge of pneumococcal physiology and response to a commonly used drug. To unveil pneumococcal adaptation to a commonly used drug, paracetamol, pneumococci were grown in CDM in the presence of 5 mM paracetamol. The response of strain D39 to paracetamol is complex, comprising a network of genes involved in fatty acid metabolism and biosynthesis, nutrition and waste transport, environmental stress detection, and antibiotic production.

Fatty acid biosynthesis and metabolism gene cluster was significantly downregulated in the presence of paracetamol. The antibacterial effect of fatty acids on the potential of S. pneumoniae to induce disease is poorly understood. Fatty acid synthase system type II (FASII) manufactures pneumococcal fatty acids which are needed for the cell membrane (Zhang and Rock, 2008), which is encoded by the fab gene cluster. This cluster's transcription is regulated by the MarR-type transcriptional regulator FabT, which suppresses the cluster responsible for fab-transcription when fatty acids attach to it (Jerga and Rock, 2009). The pneumococcus can integrate fatty acids that are produced outside (exogenous) into its membrane via the FakA/B system, in complement to de novo synthesis by the FASII system (Parsons et al., 2015). Exogenous fatty acids have been shown to reduce pneumococcal colonization in investigations, while the molecular mechanism for this antibacterial activity of free fatty acids is unknown (Bomar et al., 2016). The distinct pneumococcal SpFakB3 can be used due to its special polyunsaturates. To bind the fatty acid carbonyl and normalize the protein, pneumococcal FakB3 uses a distinct hydrogen bond network than other FakBs (Gullett et al., 2019). Deletion of fakB3 in S. pneumoniae strain JMG1 led to reduction in linoleate incorporation from human serum confirms the significance of fakB3 in this process. FakB3 (spd-0646) was one of the genes that was downregulated in our transcriptomic analysis in the presence of paracetamol, which might suggest that fakB3can be a very important target for paracetamol and further studies will be needed for deep research. Moreover, the fab genes were shown to be downregulated in pneumococcus in the presence of penicillin (Rogers et al., 2007). These genes may share a stress response to cell wall inhibitors since they respond similarly to paracetamol and vancomycin, with some of them potentially important in shielding the cell from their effects.

Iron has a pivotal role in the pathogenesis of S. pneumoniae. To successfully support infections and survival, pneumococcus has diversified three transporters named ABC, PiuABC, PiaABC, and PitABC, with lipoproteins PiuA PiaA, and PitA as proteins that binds to substrate to uptake iron (Yang et al., 2016). In our microarray findings, piuABC was downregulated in the existance of paracetamol. These findings were in contrast to a gene expression analysis based on microarray in S. pneumoniae which demosnstarted that the fluoroquinolone levofloxacin induced an positive regulation of the piuABC operon (Ferrándiz and de la Campa, 2014). They further suggested that upregulation of piuABC would cause a rise in intracellular iron, which would then activate the Fenton reaction, resulting in an increase in reactive oxygen species (Ferrándiz and de la Campa, 2014).

Genes (glnRA and glnPQ) involved in glutamine synthesis and uptake were among the downregulated ones in the presence of paracetamol. Penicillin therapy increases intracellular glutamine concentrations, according to a recent study (El Khoury et al., 2017). When culture media was supplemented with glutamine, it provided protection against penicillin (El Khoury et al., 2017). The glnA-encoded glutamine synthetase catalyzes the conversion of ammonium and glutamate into glutamine, and its chemical inhibition by the l-methionine sulfoximine (inhibitor) has been demonstrated to make S. pneumoniae susceptible to penicillin, even in penicillin-resistant clinical isolates (El Khoury et al., 2017). Therefore, we believe that paracetamol (by altering glutamine genes express) Interacts with glutamine metabolism, implying techniques that might be employed in standard treatment or to reverse resistance in future.

A couple of sugar systems manLMN and malPQ were also expressed in numerous ways in the presence of paracetamol. ManLMN is typically a major glucose transporter that has alos the ability of transporting a varying number of other carbohydrate substrates including mannose, fructose, galactose and N-acetyl glucosamine (Bidossi et al.,). manLMN is repressed by both the CcpA and CiaR, the response regulator of the conserved TCS CiaRH implicated in competence, autolysis and β-lactam resistance (Halfmann et al., 2007; Carvalho et al., 2011). In S. pneumoniae D39, inactivation of manM encoding the PTS EIIC component, resulted in a mild growth defect in glucose, and more severely reduced growth in N-acetyl glucosamine, mannose, and galactose (Bidossi et al.,). In contrast to D39, ManLMN was found to be essential for growth on five non-preferred carbohydrates in TIGR4, and required to induce expression of downstream metabolic genes (Fleming and Camilli, 2016). malPQ are the maltose utilization genes and have been shown to be regulated by MalR (Afzal et al., 2015). They code for a maltodextrin phosphorylase and a 4-alpha-glucanotransferase, respectively. These maltose genes have been demonstrated to be positively regulated during involvement of cellobiose as well (Shafeeq et al., 2013). Our β-galactosidase assays with PmalP-lacZ in the presence of cellobiose showed that the activity of PmalP was significantly higher in the presence of cellobiose as compared that in glucose (data not shown). This might indicate about the complexity of the role of malPQ in the life-style of pneumococcus and differential expression of malPQ in the presence of paracetamol corroborates our observation. Moreover, both these manLMN and malPQ systems have been shown to be differentially expressed in the presence of penicillin and vancomycin in S. pneumoniae (Rogers et al., 2007; Haas et al., 2004).

We discovered genes of S. pneumoniae that are differently expressed in reaction to paracetamol exposures in our investigation. Several of these genes have been connected to drug resistance or tolerance in the past, demonstrating that their altered expression is part of a stress-protective response in this situation. Such genes could be used as therapeutic targets to improve the effectiveness of paracetamol against this pathogen. Other gene expression changes discovered here could also point to potential paracetamol resistance mechanisms. In this context, more research on these genes is required.