Meningitis pathogens evade immune responses by thermosensing

Bacterial meningitis is a major cause of death and disability in children worldwide. Two human restricted pathogens, Streptococcus pneumoniae and Haemophilus influenzae, are the major causative agents of bacterial meningitis, attributing to 200,000 deaths annually. These pathogens are often part of the nasopharyngeal microflora of healthy carriers. However, what factors elicit them to disseminate and cause invasive diseases remain unknown. Elevated temperature and fever are hallmarks of inflammation triggered by infections and can act as warning signal to these pathogens. Here, we investigate whether these pathogens could sense environmental temperature to evade host complement-mediated killing. We show that expression of two vital virulence factors and vaccine components, the capsule and factor H binding proteins, are temperature dependent. We identify and characterize four novel RNA thermosensors in S. pneumoniae and H. influenzae within their 5′-untranslated regions of genes, responsible for capsular biosynthesis and production of factor H binding proteins. Our data further demonstrate that these pathogens have co-evolved thermosensing abilities independently with unique RNA sequences, but distinct secondary structures, to evade the human immune system. Author Summary Streptococcus pneumoniae and Haemophilus influenzae are bacteria that reside in the upper respiratory tract. This harmless colonization may progress to severe and often lethal septicaemia and meningitis, but molecular mechanisms that control why these pathogens invade the circulatory system remain largely unknown. Here we show that both S. pneumoniae and H. influenzae can evade complement killing by sensing the temperature of the host. We identify and characterize four novel RNA thermosensors in S. pneumoniae and H. influenzae within their respective 5′-untranslated regions of genes, influencing capsular biosynthesis and production of factor H binding proteins. Moreover, we show that these RNA thermosensors evolved independently with exclusive unique RNA sequences to sense the temperature in the nasopharynx and in other body sites to avoid immune killing. Our finding that regulatory RNA senses temperatures and directly regulate expression of two important virulence factors and vaccine components of S. pneumoniae and H. influenzae, is most important for our understanding of bacterial pathogenesis and for vaccine development. Our work could pave the way for similar studies in other important bacterial pathogens and enables clinicians and microbiologists to adjust their diagnostic techniques, and treatments to best fit the condition of the patients.


Abstract
Bacterial meningitis is a major cause of death and disability in children worldwide. Two human restricted pathogens, Streptococcus pneumoniae and Haemophilus influenzae, are the major causative agents of bacterial meningitis, attributing to 200,000 deaths annually. These pathogens are often part of the nasopharyngeal microflora of healthy carriers. However, what factors elicit them to disseminate and cause invasive diseases remain unknown. Elevated temperature and fever are hallmarks of inflammation triggered by infections and can act as warning signal to these pathogens. Here, we investigate whether these pathogens could sense environmental temperature to evade host complement-mediated killing. We show that expression of two vital virulence factors and vaccine components, the capsule and factor H binding proteins, are temperature dependent. We identify and characterize four novel RNA thermosensors in S. pneumoniae and H. influenzae within their 5´-untranslated regions of genes, responsible for capsular biosynthesis and production of factor H binding proteins. Our data further demonstrate that these pathogens have co-evolved thermosensing abilities independently with unique RNA sequences, but distinct secondary structures, to evade the human immune system.

Introduction
Streptococcus pneumoniae and Haemophilus influenzae are both human restricted pathogens that may cause deadly meningitis and sepsis. In order to survive in the host, these bacteria have evolved analogous survival mechanisms such as the production of polysaccharide capsules to evade host immune responses. Encapsulated bacterial pathogens pose a major threat to human health and contribute to significant morbidity and mortality [1]. For Gram-positive pneumococci, the capsule enables the bacteria to avoid phagocytosis, and at least 97 different capsular serotypes have been identified as of 2015 [2]. Pneumococci have been suggested to be resistant to complement killing due to their thick layer of peptidoglycan [3]. Gram-negative H.
influenzae shows six capsular serotypes, facilitating the bacterium to resist phagocytosis and complement-mediated killing [4]. Vaccines based on bacterial polysaccharide capsules are being used to prevent S. pneumoniae and H. influenzae infections [5].
In addition to expression of capsules, these two pathogens also evade complementmediated killing by binding to human Factor H (FH), the major negative regulator of the alternative complement pathway. FH is recruited by high affinity interactions with FH binding proteins expressed on the bacterial surface, such as Pneumococcal Surface Protein C (PspC) and the Protein H (PH, encoded by the lph gene) for H. influenzae [6][7][8]. FH binding proteins have been included in a vaccine against Neisseria meningitidis, but have also been suggested as vaccine candidates against pneumococcal infections [9][10][11].
The ecological niche for S. pneumoniae and H. influenzae is the nasopharyngeal cavity from where they may spread into the bloodstream and cause disease. There is a temperature gradient in the nasal cavity where these two pathogens colonize. The temperature on the surface of the anterior nares is around 30°C to 32°C at the end of inspiration, and rises to around 34°C in the posterior nasopharynx and tonsillar region [12,13]. Both these sites on the mucosal surface are significantly cooler than the core body temperature of 37°C, where the bacteria replicate during invasive diseases. Previously, we showed that temperature acts as a danger signal to N. meningitidis, prompting the bacterium to enhance expression of immune evasion factors via three independent RNA thermosensors [14]. RNA thermosensors are elements usually located in the 5´-untranslated region (5´-UTR) of an mRNA transcript, forming a secondary structure at lower temperatures that inhibits protein translation by blocking access of ribosomes to the ribosome binding site (RBS). As temperature rises, the RNA secondary structure undergoes a conformational change due to higher thermodynamic energy, exposing the RBS, and thus allowing translation [15].
The importance of temperature sensing in meningococci prompted us to investigate whether temperature affects expression of virulence factors in other meningitis causing pathogens that colonize the same niche, i.e. S. pneumoniae and H. influenzae. Although the role of the capsule and FH binding proteins in S. pneumoniae and H. influenzae in complement evasion has been studied, their regulatory mechanisms remain largely unknown. Here we identify that thermosensing governs the expression of the capsular polysaccharide and FH binding proteins in S. pneumoniae and H. influenzae, thereby influencing complementmediated evasion. Moreover, through sequence analyses of their 5´-UTRs, we found that the three meningitis causing pathogens have independently evolved unique RNA sequences, with no sequence conservation, to serve the same thermosensing function.

Results
The pneumococcal capsular polysaccharide is encoded by a cluster of 10-20 tightlylinked genes [16]. The first four genes, that are located at the 5′-end of the capsular locus (cpsABCD), are common to all serotypes and are involved in the capsular regulation [17]. To investigate whether temperature is involved in capsular gene expression, we examined the expression of capsular polysaccharide synthesis protein B (CpsB) in the TIGR4 strain (of serotype 4) [18]. The TIGR4 strain was grown at three temperatures: 30°C, 37°C and 42°C with no observable growth defect. The expression of the CpsB protein from total protein lysates was consistently higher at elevated temperatures in three independent experiments ( Fig 1A). As CpsA is co-transcribed and translated with CpsB, subsequent experiments were performed using CpsA. Two other pneumococcal proteins, not affected by temperatures, were used as controls (the autolysin LytA, and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) ( Fig   1A).  RNA thermosensors are elements located within specific 5´-UTR of mRNAs and they could function in a heterologous host [14,20]. Therefore, the whole intergenic regions upstream of cpsA and bcsA together with their respective first 24 coding-bases were translationally fused to a green fluorescent protein within the plasmid pEGFP-N2 and transformed into Escherichia coli. Thermoregulation of CpsA-GFP and BcsA-GFP was evident also in E. coli ( Fig 1C). RecA antibody (Abcam) was used as a cytoplasmic loading control and anti-Neomycin-Phosphotransferase antibody (NovusBio) to eliminate potential effects of temperature on plasmid replication (Fig 1C). The two previously identified RNA thermosensors, the neisserial capsular CssA and the listerial transcriptional activator PrfA, were used as positive controls (S1C Fig). In addition, site-directed mutageneses were formed by inactivating either promoter- Western blot analysis showed that both promoters of pneumococcal cps are required for production of Cps, however, only the longer transcript regulated by the novel promoter  influenzae.
Using Vienna RNAfold package [21] and VARNA applet [22], we determined the putative secondary structure of cpsA and bcsA 5´-UTRs together with their first 24-coding bases. Expression of CpsA-GFP from the plasmids containing these changes was consistent with a thermosensor in the 5´-UTR ( Fig 1E).
An elevated expression of the CpsA/CpsB proteins is however not a definite evidence that more capsule is produced. To address this, the production of capsular polysaccharides of S. pneumoniae was analyzed using a dot blot assay, as it has previously been used to quantify pneumococcal capsule production [23]. Dot blots using serotype 4 specific anti-sera showed that the polysaccharide capsule was produced in higher amounts with increasing temperature in the bacterial supernatant ( Fig 1F).
To investigate whether other pneumococcal capsules could regulate the expression of their capsular genes by sensing temperature, three pneumococcal strains of important serotypes, 1, 2, and 22F, were studied. Thermoregulation of CpsB was evident also in these strains (S6A We then examined whether other factors involved in immune evasion are subjected to similar thermoregulation and focused on FH binding proteins. Using an antibody recognizing the FH binding pneumococcal protein PspC, we observed that expression of PspC is also temperature dependent in different pneumococcal strains (Fig 2A and S6A Fig). Likewise, we also found that expression of the H. influenzae FH binding protein PH and its ability to bind to FH is also temperature dependent (  influenzae. RecA is used as loading control. C) Fluorescent flow cytometry shows increased human FH binding to S. pneumoniae when grown at higher temperatures (histograph). Fluorescence intensity of three experiments was pooled and FH binding of S. pneumoniae grown at 40°C compared to 32°C is shown (bar graph). D) Fluorescent flow cytometry shows two populations of H. influenzae with increased FH binding when grown at 40°C (histograph). E) Fluorescent microscopy analysis reveals that bacteria bind more FH at 40°C (more banded pattern of FH (green) binding to S. pneumoniae (auto-fluorescence of bacteria in red)) than at 32°C. Bar = 10µm.F) Fluorescent microscopy analysis reveals a FH (green) positive population of H. influenzae (auto-fluorescence in red) along with a FH negative population at 40°C. Bar = 10µm. Error bars denote s.e.m. * p < 0.05, ** p < 0.01 (Student's t-test).
The finding of an elevated factor H binding proteins at higher temperatures from protein lysates are not definite evidence that more surface exposed proteins are produced, nor that FH binding occurs to the bacterial surface. To establish whether temperature could affect binding of FH, S. pneumoniae and H. influenzae grown at different temperatures were exposed to fluorescently labeled FH and analyzed by flow cytometry. S. pneumoniae showed an increased ability to bind FH when grown at 40°C, compared to 32°C (Fig 2C). Fluorescence microscopy staining was consistent in showing higher intensity of FH binding in S. pneumoniae grown at 40°C compared to 32°C (Fig 2E). Similarly, H. influenzae grown at different temperatures also showed differences in the flow cytometry analysis (Fig 2D). Two populations were observed when grown at 40°C as indicated by the two maxima in the histogram. When exposed to labeled FH, bacteria from this culture showed a clear increase in fluorescence intensity. Fluorescence microscopy staining revealed almost no fluorescence on bacteria grown at 32°C. When grown at 40°C, a subpopulation was found that was strongly positive for FH, while remaining bacteria were negative (Fig 2F). The microscopy observation is consistent with the two maxima in flow cytometry.
To test if higher expression of polysaccharide capsules and FH binding proteins at higher temperature could increase bacterial survival when facing human immune factors, serum killing and opsonophagocytosis assays were performed. H. influenzae was grown overnight at 30°C and pre-incubated at 30°C or 37°C for one hour prior to serum killing assays. H. influenzae pre-incubated at 37°C showed a significantly higher serum survival rate after 30 minutes compared to those at 30°C (Fig 3A). Pneumococci are naturally resistant to serum killing, therefore the protective role at different temperatures was studied using phagocytosis assays. S. pneumoniae was grown at either 30°C or 37°C, and opsonized with human serum prior to incubation with human THP-1 macrophages. Serum opsonized S. pneumoniae grown at 37°C was phagocytosed significantly less than those grown at 30°C (Fig 3B). Altogether the results show that at higher temperature, both S. pneumoniae and H. influenzae are better at evading complement mediated killing.

Figure 3
Temperature changes influence complement escape by S. pneumoniae and H. influenzae. A) H. influenzae were pre-incubated at 30°C or 37°C in RMPI for 1 hour prior to addition of human serum. The mixtures were then incubated at 37°C for 30 minutes. H. influenzae pre-incubated at 37°C shows more resistance to complement-mediated killing than those at 30°C. B) Opsonophagocytosis assay: S. pneumoniae TIGR4 was grown at 30°C or 37°C. The bacteria were then opsonized with human serum for 30min at 37°C. Non-opsonized bacteria were exposed to only RPMI for 30min at 37°C. Pneumococci grown at 37°C are more resistant to phagocytosis by macrophages than those grown at 30°C. Experiments were performed on three separate occasions and error bars denote s.e.m. * p < 0.05, ** p < 0.01 (Student's t-test). C) Model of pathogenesis, from commensalism to systemic infection. Infection or other factors induce mucosal surface inflammation and a raise in temperature, leading to increased expression of virulence determinants such as capsule and FH binding proteins of S. pneumoniae, H. influenzae and N. meningitidis. This thermoregulation helps the bacteria to evade immune responses and enables better bacterial survival. The compromised integrity of the mucosal surfaces during viral replication serves as site of dissemination for bacteria into the circulatory system. Consequently, the temperature adapted bacteria will have better chance of surviving within warmer body sites, increasing the rate of systemic infections. Elevation of temperature has been shown to act as a danger signal for N. meningitidis, prompting the bacterium to enhance immune evasion [14,27]. Biosynthesis of the meningococcal sialic acid containing capsules and fHbp is governed by two independent RNA thermosensors. In agreement with this finding, we here show that S. pneumoniae and H.

Discussion
influenzae also are able to evade complement mediated killing by sensing temperatures via RNA molecules.
Through molecular and biochemical analyses of S. pneumoniae and H. influenzae, we identified four sequence unique RNA thermosensors controlling the expression of their capsular gene (Cps and Bcs) and FH binding proteins (PspC and PH). Previous studies have shown that mutations within the 5´-region of cps could disrupt the production of the capsule [23,28].
However, these authors did not fully understand mechanisms leading to disruption of the capsule production. Our findings here explain that the mutations generated would stabilize the RNA thermosensor's secondary structure thus resulting in lower capsule production. Like all other characterized RNA thermosensors [14,20], we were able to show that single nucleotide mutations in the cps 5´-UTR could disrupt its thermosensing abilities. Furthermore, we found that the nucleotide sequences within the 5´-UTR of cps, essential for forming the RNA secondary structure, are highly conserved among various pneumococcal strains of different capsular serotypes. Naturally occurring polymorphisms in the cps 5´-UTR of serotypes 1, 2 and 22F could neither alter the putative RNA secondary structures, nor the thermoregulation ability of Cps (Fig S6).
Our results show that the increased translation of pneumococcal CpsA at higher temperature indeed led to higher production of capsule. Studies have previously shown that capsular shedding enable pneumococci to evade complement killing and facilitate bacterial spread [29,30]. These shed capsules are immunogenic and free floating components that could additionally sequester antibodies and immune cells. In agreement with these studies, we also observed more capsule in the supernatant from pneumococci grown at higher temperature.

Temperature mediated production of capsules and binding of human FH in S.
pneumoniae and H. influenzae could indicate better protection for the bacteria from immune killing. Our work here demonstrates that both bacteria are indeed able to evade complement mediated killing at higher temperature as shown using assays for serum killing and opsonophagocytosis. An increase in temperature triggered by inflammation in the host may act as a 'danger signal' for S. pneumoniae and H. influenzae priming their defenses against the recruitment of immune effectors onto the mucosal surface. While attuned to higher threat, it remains largely unknown how these pathogens breach from the mucosa into the bloodstream and further into the brain. We hypothesize that the nasopharyngeal tissue could be damaged during prior Influenza A viral infections, serving as an entry site for the bacteria. Temperature increment caused by a local inflammation by primed pathogens could enhance evasion of immune responses and increase bacterial growth, leading to bacteremia and the pathogen crossing the blood brain barrier to cause meningitis [31][32][33]. A proposed infection model influenced by temperature is shown in Fig 3C. The expression of capsular polysaccharides and FH binding proteins is an important survival strategy for these meningitis causing pathogens. However, overexpression of such factors at certain sites, such as on nasopharyngeal mucosal surfaces, could evoke undesired immune reactions for the bacteria, thus jeopardizing colonization. It has previously been demonstrated that high capsular expression in pneumococci may have negative effects on adhesion to the respiratory mucosa [34]. Thermosensor mediated modulation of capsular expression may therefore be a central strategy for these meningitis pathogens to optimally colonize their normal habitat, the human nasopharynx. At the same time, and when necessary (i.e. local inflammation), these bacteria could rapidly express certain properties in order to avoid immune killing.
We here reveal that human restricted nasopharyngeal pathogens, S. pneumoniae and H.
influenzae, sense ambient temperature changes. RNA thermosensors positively influence the expression of their major virulence determinants at warmer growth conditions. Interestingly, the four novel RNA thermosensors described here, together with the two known neisserial RNA thermosensors, do not possess any sequence similarity among them, but all retain the same thermosensing ability. This suggests that while nucleotide sequences within the 5´-UTR could be dispensable, a functional RNA thermosensor imperatively depends on its secondary structure with a weakly base-paired RBS. It is most likely that these RNA thermosensors in S. pneumoniae, H. influenzae and N. meningitidis have evolved independently to sense the same temperature cue in the nasopharyngeal niche to avoid immune killing.

RNA-mediated virulence gene regulation is still an understudied phenomenon.
However, with recent RNA sequencing methods such as RNA structurome sequencing analysis [35], comprehensive RNA thermosensor maps could be generated. In addition, nuclear magnetic resonance (NMR) spectroscopy could be used to investigate the RNA structures [36][37][38]. We believe that such studies will provide valuable information on the functional importance and dynamics on RNA regulation in these pathogens, ultimately contributing to the understanding of bacterial pathogenesis.

Bacterial strains, plasmids and growth conditions.
Strains, oligonucleotides and plasmids are listed in Table 1 The resulting constructs were sequenced to ensure that no other changes had occurred prior transformation into XL10-Gold E.coli (Agilent). Table 1 Oligonucleotides, strains and plasmids

Site-Directed Mutagenesis
In order to obtain site-specific mutations in CpsA-GFP constructs, we used the Phusion High-Fidelity DNA Polymerases (Thermo Scientific) for site-directed mutagenesis. A plasmid harboring the CpsA-GFP fragment was used as template in the mutagenesis reactions. To achieve disrupted thermosensing, CpsA mut-1 and mut-2, oligonucleotide pairs of Mut1-F with Mut1-R and Mut2-F with Mut2-R respectively, were used (Table 1) Table 1.  C) Thermoregulation of CssA-and PrfA-GFP fusion protein is detected in E. coli by western blot analysis. RecA and Neomycin-phosphotransferase (Npt) are used as loading controls.
B) Western blot analysis shows that thermoregulation of CpsA is disrupted in a Promoter-1 mutant (Prom-1 Mut) whereas a mutant in Promoter-2 (Prom-2 Mut) retains its thermosensing ability. Site-directed mutageneses were performed to replace the -35 sequences of each promoter. In Prom-1 Mut background, only promoter 2 is active and in the Prom-2 Mut background, only promoter 1 is active. RecA was used as a loading control.

S4 Fig.
In vitro transcription/translation assay comparing capsule producing enzymes with known thermosensors at a range of biological temperatures. CpsA and BcsA show thermosensing. N. meningitidis CssA and L. monocytogenes PrfA thermosensor are used as positive controls. Blots are representative of experiments performed on at least three occasions.