- Research article
- Open Access
Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages
Veterinary Research volume 47, Article number: 13 (2016)
Streptococcus uberis is frequently isolated from the mammary gland of dairy cattle. Infection with some strains can induce mild subclinical inflammation whilst others induce severe inflammation and clinical mastitis. We compared here the inflammatory response of primary cultures of bovine mammary epithelial cells (pbMEC) towards S. uberis strains collected from clinical or subclinical cases (seven strains each) of mastitis with the strong response elicited by Escherichia coli. Neither heat inactivated nor live S. uberis induced the expression of 10 key immune genes (including TNF, IL1B, IL6). The widely used virulent strain 0140J and the avirulent strain, EF20 elicited similar responses; as did mutants defective in capsule (hasA) or biofilm formation (sub0538 and sub0539). Streptococcus uberis failed to activate NF-κB in pbMEC or TLR2 in HEK293 cells, indicating that S. uberis particles did not induce any TLR-signaling in MEC. However, preparations of lipoteichoic acid (LTA) from two strains strongly induced immune gene expression and activated NF-κB in pbMEC, without the involvement of TLR2. The immune-stimulatory LTA must be arranged in the intact S. uberis such that it is unrecognizable by the relevant pathogen receptors of the MEC. The absence of immune recognition is specific for MEC, since the same S. uberis preparations strongly induced immune gene expression and NF-κB activity in the murine macrophage model cell RAW264.7. Hence, the sluggish immune response of MEC and not of professional immune cells to this pathogen may aid establishment of the often encountered belated and subclinical phenotype of S. uberis mastitis.
The Gram-positive bacterium Streptococcus uberis is among the four most prevalent species of mastitis causing pathogens [1, 2]. Infection with this bacterium can occur with very few if any clinical signs, but can also result in severe inflammation of the udder culminating in clinical mastitis . It is not entirely clear if the heterogeneous physiology of S. uberis mastitis is caused by genetic diversity of the different strains infecting the udders. There is an extraordinary diversity of S. uberis strains [4, 5] and clear examples of strains that reproducibly induce clinical or subclinical infections have been reported [5–7]. There are reports that occasionally a predominant strain infected several cows within a herd  but it was also found that genetically distinct S. uberis strains infected different individuals within a herd and distinct strains caused re-infection after a previous successful cure of a first S. uberis infection [9, 10].
Multilocus sequence typing studies suggested that mastitis causing S. uberis strains (clinical and subclinical) may be genetically different from avirulent strains . However, no clear gene-loss or -gain correlation with the virulent or avirulent phenotype of the strains emerged in a very recent comparison of the whole genome sequences from thirteen different S. uberis strains . This suggests that the particular outcome of an udder infection is largely determined by the host-pathogen interaction rather than by the particular genotype of the pathogen.
Streptococcus uberis vs. cow interactions have been studied in several udder infection experiments. S. uberis generally elicited a belated onset of inflammation, compared to infections with E. coli or other Gram-negative pathogens [12, 13]. Comparing global transcriptome profiling from S. uberis vs. E. coli infection trials reveals a remarkable failure of S. uberis to induce expression of pro-inflammatory cytokine and chemokine-encoding genes in the udder [14–19]. It rather appeared that S. uberis infection up-regulated IL10 and IL6 governed pathways, which are both known to eventually counteract strong inflammation . Moreover, these studies recapitulated the physiological variability in the outcome of S. uberis udder infections. While the widely used strain 0140J—known as the almost prototypical strain for eliciting clinical mastitis [6, 20]—caused clinical mastitis in one of these infections trials , it elicited subclinical mastitis in the second trial .
Mammary epithelial cells (MEC) of the lactating udder parenchyma are the dominant cell type coming into contact with invading pathogens early on after infection. Quantitative morphometry revealed that MEC comprise more than 70% of the udder cells [21, 22]. MEC are the dominant sentinels of the lactating parenchyma and competent to mount the first cytokine alert [17, 23–25]. They express not only the relevant Toll-like-receptors (TLRs) for perceiving pathogens but also β-defensin-encoding genes to counteract alveolar colonization of the pathogens [26–28]. It was reported that challenging these cells with only some strains of S. uberis, but not with others, would activate cytokine genes expression in these cells [14, 29].
Epithelial cells are known to perceive pathogens through pathogen recognition receptors (PRR) including the family of TLRs . Thirteen mammalian TLRs are known. Binding of their ligands (collectively known as Pathogen Associated Molecular Patterns; PAMPs) activates their downstream signaling. This ultimately leads to the activation of the NF-κB factor complex, mediated through a multifactorial cascade . These transcription factors regulate the expression of a wealth of immune genes [32–34]. TLR2 is known to be essential for mounting an efficient immune defence against Gram-positive bacteria [35–37]. Lipoproteins from these pathogens belong to its natural ligands [38, 39]. However, it was reported that Gram-positive Group B streptococci (e.g. S. agalactiae) did not activate TLR2 . Moreover, mitomycin-C inactivated S. uberis preparations did not activate the bovine TLR2 receptor in HEK293 cells . Failure to activating a TLR mediated immune response would readily explain the slow and weak immune response caused by a S. uberis challenge.
We wanted to know if the divergent physiology of S. uberis mastitis might in tendency be related to S. uberis strain specific differences in the interaction with the MEC. Therefore, we wanted to establish a broader survey of the capacity of diverse S. uberis isolates from clinical and subclinical cases of mastitis to stimulate immune functions of MEC. We included previously used model strains either known to causing mastitis or as being avirulent for direct comparison of the results with current knowledge. We were also curious about the possible divergent immune stimulatory properties of cell wall components isolated from different strains to see if they would possibly cause a strain dependent difference in eliciting an immune response.
Our parameters for the induction of immune functions consisted of the pathogen challenge -related modulation of the expression of a panel of cytokine- and chemokine-encoding genes (such as TNF, IL1A, IL6, CXCL8) but also those encoding effectors of immune defence (β-defensin LAP, NOS2A) or membrane protecting factors (SAA). Their relevance as markers for induced immune functions in MEC has previously been established . Additionally, we monitored the activation of the NF-κB transcription factor complex. We show that—under our experimental conditions—all the S. uberis strains failed to significantly induce immune functions in the MEC, but not in macrophage model cells.
Materials and methods
Mastitis pathogen strains
Streptococcus uberis strains used in this study are 233 (obtained from the AgResearch New Zealand ), 0140J (ATCC® Number BAA-854, isolated from clinical mastitis, 1972 in the UK), EF20 (avirulent strain isolated from clinical mastitis, 1970 in the UK , ΔhasA (noncapsular hyaluronate synthase A mutant derivative of strain 0140J; ); 0140J::ISS1 P’ (mutant of 0140J, inactivation of the promoter addressing the glycosyltransferase genes sub0538 and sub0539 which significantly reduced the ability to form biofilm ); T1–18 and T2–58 (isolated from cases of mastitis in the UK, provided by Dr Michael Fontaine, Moredun Research Institute, UK). The S. uberis strains C6344, C5072, S6261, C9359, Ab71 (all isolated 2002 in the UK), 4428 (1999, UK), 5291 (2000, UK) were isolated from clinical cases while C8329, C5388, C7131, S7010 (all isolated 2002 in the UK), B190, B362 (2000, UK), 6736 (1999, UK) were isolated from subclinical cases of mastitis. S. agalactiae 0250 and S. dysgalactiae 2023 were isolated from cases of bovine mastitis in the UK and are included in the National Institute for Research in Dairying culture collection (currently hosted by Prof. James Leigh at the University of Nottingham). E. coli strain 1303 is a well characterized mastitis model strain isolated from udder secretions of a cow with clinical mastitis .
Bacterial growth and preparation of pathogen particles
Details regarding culturing E. coli 1303 and its use to challenge the pbMEC were exactly as described . S. uberis, S. agalactiae and S. dysgalactiae were grown in Todd Hewitt Broth (THB, Carl Roth GmbH) at 37 °C without agitation to the logarithmic phase of culture growth (0.5, OD600 nm). Plating of dilution series was used to calibrate cell counts from the OD readings. Efficacy of killing the bacteria through heat treatment (60 min, 80 °C) was verified by control plating. Heat treated cells were collected by centrifugation, washed twice with RPMI 1640 medium (Biochrom), and re-suspended therein. Aliquots were stored frozen at −20 °C. We applied in challenge experiments similar protein concentrations of the heat killed bacteria from the different strains in order to standardize the conditions. Protein contents of the bacterial preparations had been determined with the Lowry procedure . Based on three independent growth experiments, we found from exponentially multiplying cultures (OD600nm, 0.5) as protein content ~16.8 ± 4.1 and ~5.8 ± 0.8 µg/107 bacteria for of E. coli 1303 and S. uberis strain 0140J, respectively. The other S. uberis strains had protein contents similar that determined for 0140J.
LTAs were isolated as described by Morath et al.  with few modifications as detailed . Treatment of lyophilized native LTA with 1% H2O2 in phosphate-buffered saline (PBS) inactivated contaminating lipoproteins . Our NRM spectroscopy analysis proved that the LTA core structure was not altered by that treatment. LTA preparations were endotoxin free as judged by their failure to activate the bovine TLR4 in the HEK293 cells. The latter assays were conducted as previously described .
Cell culture procedure
Tissue cultures of pbMEC were established as described . Their cultivation and pathogen stimulation on collagen IV coated tissue culture plates in RPMI 1640 (Biochrom) supplemented with insulin, prolactin, dexamethasone and 10% FCS (PAN-Biotech) was as detailed by Günther et al. . Briefly, frozen aliquots of pbMEC were seeded at high cell density into 9 cm dishes, purified through selective trypsinization and reseeded for experiments into six well plates, again at high cell density. After settling overnight duplicate wells were challenged at various times (t0, t21, t23 h) through the addition of 30 µg/mL of protein from heat killed bacteria. Duplicate unstimulated control cultures were kept in parallel. All cultures were collected at t24 h for RNA extraction.
RAW264.7 cells (from ATCC) were cultivated in DMEM (Biochrom) supplemented with 2 mM l-glutamine and 10% FCS. Stimulation experiments were similarly performed using 80% confluent cell cultures.
All tissues and cells were retrieved from healthy first lactating Holstein–Friesian heifers having been slaughtered in our local abattoir, complying with all pertinent ethical and legal requirements. The abattoir is a EU licensed (ES1635) core facility of the research affiliation and serves to routinely supply samples to different laboratories. Special ethical approval was unnecessary since the cows had been culled in the normal culling regime without conducting any animal experimentation.
RNA extraction and mRNA quantification
RNA was extracted with TRIZOL-reagent (Invitrogen). Preparation of the cDNA (Superscript II, Invitrogen) and real time quantification of the mRNA concentrations with the Fast-Start Sybr Green I kit and the LightCycler II instrument (Roche) were done essentially as described , however using per assay a cDNA input derived from 75 ng of total RNA. Titration of relative copy numbers against external standards and normalization against the not regulated reference gene chloride intracellular channel 1 (CLIC1) were done as detailed in . Sequences of oligo nucleotide primers are listed in Additional file 1.
Determination of NF-κB activation
NF-κB activity was assessed with a reporter gene expressing the Renilla-luciferase under the control of the NF-κB activated ELAM promoter (Invivogen; ). This reference describes also the vector expressing the bovine TLR2 receptor. These constructs were transfected into pbMEC and HEK293 cells with Lipofectamine 2000 (Invitrogen) essentially as previously described in detail .
Macrophages are notorious for being difficult to transfect due to the natural response of phagocytes against foreign materials. Therefore RAW264.7 cells were transfected using the Neon® Transfection System (Life Technologies) following the manufacturer’s instructions for this specific cell type. Briefly 5 µg of the ELAM NF-κB reporter plasmid were used to transfect 106 cells with one pulse of 1580 V for 20 ms. Subsequently the cells were seeded into wells of 24-well plates. The cells were allowed to recover overnight prior to stimulation. After challenging with the respective stimulus for the time as indicated, the cells were lysed and luciferase activity was assayed using the dual luciferase assay reporter system (Promega) as detailed . The enzyme activity was calibrated against the protein content of the lysate.
Stimulation with live pathogens
Pathogens were grown to the logarithmic phase of culture growth (0.5, OD600nm) in the respective growth medium. Subsequently, the bacteria were washed twice with RPMI 1640 medium and resuspended therein. They were co-cultured with pbMEC in RPMI 1640 medium (without antibiotics) for 1 h with 107 CFU/mL of the respective pathogen. Subsequently pathogens were killed by adding 100 µg/mL gentamicin. For mRNA quantification the pbMEC were either instantly harvested (1 h time point) or cultured for another 2 or 23 h in pbMEC growth medium (3 and 24 h time point, respectively). For luciferase measurement of NF-κB activation lysates were prepared 23 h after bacterial killing with gentamicin.
The data were analysed with GraphPad Prism Version 5 (GraphPad Software, Inc., La Jolla, CA, USA). Differences were evaluated through an analysis of variance (ANOVA) including Bonferroni’s correction for pairwise multiple comparisons.
Streptococcus uberis strains failed to activate immune gene expression in pbMEC
Dose finding studies had confirmed in pilot experiments that preparations of the heat-killed particles from the non-encapsulated strain 233 did not significantly induce cytokine gene expression in pbMEC , even if applied at high concentrations (up to 108 particles/mL; data not shown). The pathogen concentration in the milk of S. uberis infected udders is known to reach ~107 CFU/mL [5, 13]. We then surveyed the immune stimulatory properties of a broader collection of strains isolated from either clinical or subclinical cases of mastitis to eventually find indications for strain specific differences. pbMEC cultures were stimulated for up to 24 h with 30 µg/mL of protein from heat-killed preparations of seven different strains each isolated from clinical or subclinical cases of mastitis. Thus, approximately 100 bacterial particles were applied per MEC host cell (MOI ~100). A similarly preparation of E. coli 1303 was included as a positive control. Contrary to E. coli, all S. uberis strains failed to significantly activate gene expression of seven different immune genes (TNF, CXCL8 (Figure 1), IL1A, IL6, CCL5, SAA3, β-defensin LAP; data not shown). No difference was observed between isolates obtained from clinical or subclinical infection. However, expression of CYP1A1 was strongly induced to a similar extent by all bacterial preparations (Figure 1), validating that the cells had perceived presence of disturbing compounds in their environment. Expression of this general detoxification enzyme  is induced by a wide variety of xenobiotic stress and largely regulated by the aryl hydrocarbon receptor (AhR) and its nuclear translocator (ARNT; see [51, 52] for reviews).
We repeated the survey using another two different mastitis isolates, strain 233 and the widely used strain 0140J. Again, no significant induction of the candidate immune genes was recorded (Additional file 2) and these data validate that the absence of immune stimulatory properties of the model strains 0140J and 233 on MEC are typical for mastitis causing S. uberis pathogens.
As the presence of the capsule is positively correlated with isolates from clinical disease , we next examined if the capsule might be responsible for the poor immune stimulatory properties of S. uberis on MEC. The ΔhasA mutant of strain 0140J cannot form capsule . We found that it was as ineffective in activating an immune response in pbMEC as the wild type strain (Figure 2A). Similarly ineffective was a mutant of S. uberis strain 0140J with a fivefold reduced capacity to express a key gene involved in glycolipid formation and proven reduced capacity for biofilm formation (Figure 2B).
We have recently observed that addition of 10% FCS greatly quenched the reactivity of pbMEC against Staphylococcus aureus, another representative of a Gram-positive pathogen . Hence, we examined the effect of FCS supplementation on the pbMEC response against S. uberis. Therefore the pbMEC were grown in normal growth medium. They were then washed twice with PBS and growth medium devoid of FCS was added. The cells were challenged in this medium for three h with S. uberis 233, E.coli 1303 and similar preparations of S. aureus 1027. E. coli and S. aureus quite strongly induced the expression of most of our candidate immune genes. However, the response against S. uberis was to that recorded in normal growth medium (Additional file 3).
Streptococcus uberis did not quench the reaction of pbMEC against E. coli
It has been reported that components of the S. uberis capsule may impair neutrophil functions . We therefore examined, if pre-incubating pbMEC with S. uberis might impair the immune reactivity of the host cells. Cultures were pre-incubated for 1 h with 30 µg/mL of heat-killed particles from either of two S. uberis strains and subsequently challenged with a mild dose (3 µg/mL) of heat-killed E. coli particles. Scoring the mRNA concentrations of our panel of immune genes revealed no indication that pre-incubation reduced the immune response to E. coli; the expression of all these genes subsequent to the E. coli challenge was almost identical in S. uberis pre-incubated cultures and their respective controls (Additional file 4).
Live S. uberis elicited only a slightly enforced immune reaction
Any method to inactivate pathogens alters the surface composition of the particles. Hence, we analyzed if co-culturing the pbMEC with live pathogens would hasten and inforce the immune reaction of the pbMEC. We therefore co-cultured these cells for 1 h with 107 CFU/mL of S. uberis strain 0140J. Then the bacteria were killed through the addition of 100 µg/mL of gentamicin and the cultures were subsequently sampled at various times. Induction of immune gene expression was again found to be weaker by an order of magnitude than elicited by a similar challenge with E. coli (Table 1). However, live S. uberis pathogens induced the expression several genes (TNF, IL6, CXCL8) to a slightly larger extent than the heat-killed pathogens.
Streptococcus uberis failed to elicit TLR2 signaling and NF-κB activation in MEC
We examined if a lack of TLR activation might be the underlying cause for the absence of any immune gene activation in MEC. On the one hand we inquired about the S. uberis mediated activation of the TLR2 receptor, since there is compelling evidence that this particular TLR is crucially involved in counteracting infection by Gram-positive pathogens [35, 38] and a previous report had suggested that TLR2 might not be activated by S. uberis . HEK293 cells were co-transfected with our construct expressing the bovine TLR2 factor and the NF-κB driven luciferase reporter construct. Ligand mediated NF-κB activation would indicate TLR2 activation. None of the two S. uberis strains activated NF-κB in the HEK293 cells, even at very high concentrations of particles added (75 µg/mL equaling a MOI of approximately 300; Figure 3A). However, heat-killed particles of other streptococcal species (S. dysgalactiae and S. agalactiae) induced TLR2 to a similar extent as challenging with E. coli, which had been included as a positive control.
Pathogen-induced signaling from all TLR receptors is known to ultimately culminate in the functional activation of the NF-κB factor complex. Hence, we analyzed if S. uberis would at all activate NF-κB in pbMEC. In one set of experiments, we transfected the pbMEC cells with the NF-κB reporter construct and subsequently challenged the cells with heat-killed particles from three different stains. We found in all cases a 30% increase of the active NF-κB factors. However, the stimulations were statistically insignificant (Figure 3B, left hand panel). We repeated the experiment with live pathogens. The transfected cells were co-cultured with live pathogens (MOI, 30) for 1 h. Subsequently, the pathogens were killed (but kept in the culture fluid) by the addition of 100 µg/mL of gentamicin and the NF-κB activity was assayed 24 h later. The result was similar. Challenge with live S. uberis pathogens (strains 0140J and 233) did not significantly increase the level of active NF-κB (Figure 3B, right hand panel).
These results together show that S. uberis, either as live pathogen or as heat-killed particle avoids almost completely triggering any TLR-signaling. The particles are unrecognizable by TLR2 and this deficit is not efficiently compensated for by any other pathogen receptor.
LTAs from S. uberis activated immune gene expression and NF-κB in pbMEC, but not through TLR2 activation
Given the almost absent stimulatory capacity of the S. uberis particles, we wondered if LTAs as major cell envelope components would stimulate immune gene expression in pbMEC. LTAs were isolated from two different strains. Both preparations strongly activated expression of our candidate immune genes. The examples shown in Figure 4A and Table 2 also demonstrate that there was no difference between the LTAs isolated from the strains 233 and T1–18.
Strong induction of immune gene expression in pbMEC was accompanied by significant activation of NF-κB factors (Figure 4B, left hand panel). Inactivation of potentially co-isolated lipoprotein components through peroxide treatment significantly reduced the NF-κB stimulation by these LTA preparations. The slight residual NF-κB activation was statistically insignificant. We also investigated if other components of the S. uberis strain 233 cell envelope (lipoproteins, lipids, glycolipids) might activate NF-κB factors in pbMEC. None of those components significantly activated NF-κB in pbMEC (Additional file 5).
The NF-κB stimulatory activity of the LTA preparations was not mediated through TLR2. This became clear by analyzing the NF-κB stimulatory activity of these preparations in the HEK293 reconstitution system of TLR2 activation (Figure 4B, right hand panel). The HEK293 cells were co-transfected with the NF-κB driven luciferase reporter gene and a vector expressing the bovine TLR2 receptor. Stimulations with different dose of the native LTA preparations only slightly elevated the level of active NF-κB factors. Peroxide treatment of the same LTA preparations abolished completely their capacity to activate NF-κB. E. coli, on the other hand activated NF-κB in these experiments very strongly, by more than 11 fold.
These data together show that the cell envelope component LTA isolated from S. uberis is in principal recognizable by receptors of the pbMEC relevant for triggering an immune alert.
Streptococcus uberis strongly activated immune gene expression and NF-κB factors in murine RAW264.7 macrophage model cells
The poor immune stimulatory capacity of S. uberis was peculiar for pbMEC. This was found by stimulating the murine macrophage model cell line RAW264.7 with the same S. uberis preparations as used in the pbMEC stimulations. Challenging these cells with heat-killed particles from three different S. uberis strains resulted in a significant and strong induction of all immune genes examined (Figure 5A). They induced expression of the CXCL2 encoding gene to a similar extent as E. coli. The other three genes analyzed (TNF, IL6, CCL5) were also all significantly induced by all three S. uberis strains, albeit to a lesser extent than by E. coli. These inductions of immune gene expression were paralleled by strong and significant activation of NF-κB factors in these cells, similarly as caused by challenging them with E. coli (Figure 5B).
It is still unclear whether or not the physiological outcome of an udder infection with S. uberis as either clinical or sub-clinical mastitis relates to the divergent genotypes of respective pathogen. The controversial literature in this regard was recently extensively reviewed by Zadoks et al. . The overarching goal of our study was therefore to examine if the respective mastitis physiology might be caused by S. uberis strain specific differences in the interaction with the MEC. As a first step, we used here for most of the experiments preparations of heat killed S. uberis particles from diverse strains to ensure good technical reproducibility. It is known that PAMPs activate immune functions in MEC very quickly (<1 h; ). Our experimental setting thus mainly focused on eventual differences in the passive—PAMP-related—immune induction capacity of the various S. uberis strains and ignored the possibly very crucial effects of virulence factors secreted by the live pathogens. Their effect emerges at later times during host pathogen interaction, after their accumulation in high enough concentrations in the alveolar fluid to significantly modulate the MEC immune responsiveness.
We conducted the study on the background of our broad experiences regarding the pathogen-species specific immune response of the pbMEC model system towards challenges with E. coli and S. aureus mastitis pathogens under various experimental conditions [17, 48, 49, 54].
It is a prevalent property of S. uberis to not trigger immune functions in MEC
Our key observation is that the MEC generally does not mount an adequate immune response against S. uberis. This general immune unresponsiveness of these cells occurred in our experiments, albeit that the cells had perceived indeed the xenobiotic stress caused by the presence of the bacteria in the environment as shown by the strong, uniform and dose dependent induction of CYP1A1 expression by heat-killed bacteria, irrespective of their species. Using heat-killed preparations of 14 different strains we found (1) that none of these strains significantly induced any substantial immune gene expression and (2) no indication of any significant quantitative difference in the responses in pbMEC induced by isolates from clinical or subclinical cases of mastitis. Moreover, this uniform escape from alerting immune functions in this specific cell type of the host was independent from the capacity of S. uberis for capsule formation (0140J forms a capsule while 233 does not ; similar reaction observed against 0140J and its mutant 0140J Δhas ) or the virulent vs. avirulent phenotype of the S. uberis pathogens.
The lack of MEC responsiveness against the challenging pathogens was not due to gross surface alterations associated with the heat inactivation of the pathogens, since short-term co-culture with live pathogens and their subsequent inactivation with gentamicin conceivably left the surface structure of the pathogens unchanged and nevertheless did not cause a much stronger reaction. Gram-positive bacteria are known to evade immune recognition through masking with serum components [55–57]. However, our control experiment challenging the MEC in serum free medium proved that no such mechanism was responsible for the general unresponsiveness in the current study. The slightly enforced reactivity of the MEC against the live pathogens might be due the presence of some small RNA molecules which are found on the surface of live pathogens but which might have been washed away during the heat inactivation procedure. Such “vita-PAMPs” were found to eventually induce stronger expression of some immune genes (examples IFN-β, IL1-β) but not of others, such as IL6 . Indeed, small RNA molecules have also been found adhering to the surface of S. uberis (JA Leigh, personal communication).
Considering all these controls we can therefore exclude technical errors as cause for the observed immune unresponsiveness of the MEC against a wide variety of S. uberis strains which all had been isolated from the bovine mammary gland. However, our data are at variance with a few reports that some particular strains did indeed induce some immune reaction in pbMEC cultures [14, 29]. Such strains must be rare since we did not find a single isolate with these properties in our collection of more than twenty different strains.
Streptococcusuberis does not activate substantial TLR-signaling in MEC
We identify complete absence of any S. uberis induced TLR-signaling in MEC as a major molecular cause for the failure of these cells to mounting an immune defense against pathogens of this species. This was revealed since neither challenging the pbMEC with heat-killed nor with live pathogens increased the level of active NF-κB factors. Activation of this transcription factor complex however is the integrating indicator for PRR-signaling, including TLR- and NOD- signaling [30, 31]. We also validated in particular that S. uberis does not activate TLR2-signaling confirming previous reports . Moreover, our results demonstrate that the failure to activate PRR signaling is peculiar to S. uberis from among the streptococcal species complex. S. agalactiae and S. dysgalactiae activated the bovine TLR2 receptor in HEK293 cells. TLR2 activation through S. agalactiae was also previously reported , but has not been found by two other groups [40, 41]. No explanation can be given to resolve this discrepancy.
Purified LTA from S. uberis, but not other cell envelope components can in principle activate immune functions in MEC
We examined the immune stimulatory properties of LTA, glycolipids, lipids and lipoproteins isolated from S. uberis. Only LTA was found to significantly stimulate an immune response in MEC. LTA is an integral component of the cell envelope of Gram-positive bacteria . It is long known as having immune stimulatory properties, but it still is questionable which PRR is its cognate receptor. The controversial debate regarding the role of TLR2 for LTA recognition has recently been summarized . Our observation that isolated LTA from two different S. uberis strains strongly induced immune gene expression in MEC to similar extent is well in line with a wealth of reports showing the immune stimulatory function of such molecules [47, 61, 62]. Nuclear magnetic resonance spectroscopy experiments showed no structural differences between the LTAs isolated from strain 233 and T1-18 . Detailed structural analysis of LTA from strain 233 revealed that this LTA may be typical for streptococcal LTA, since its structure was found to be very similar to LTAs prepared from S. agalactiae and S. dysgalactiae . Significant NF-κB activation through the S. uberis derived LTA preparations in MEC strongly suggests activation of PRR-mediated signaling, but not involving TLR2 according to our data. Activation of NF-κB was associated with the integrity of the native LTA preparation. These are known to be possibly contaminated with co-purifying lipoproteins . The immune stimulatory properties of LTA and lipoproteins can be inactivated by H2O2 oxidation , and this treatment abolished any significant NF-κB activation in MEC through our LTA preparations.
Clearly, our data do not allow identification of the LTA receptor. Obviously, TLR2 is not involved in mediating the LTA elicited response. This is in line with previous reports that chemically synthesized LTA does not activate TLR2 signaling . Nevertheless, our data show that isolated cell envelope components of S. uberis may indeed strongly activate immune functions in MEC. This suggests, in turn that such immune stimulatory components are structurally arranged in the cell envelope such that they are unrecognizable to the relevant PRRs of the MEC. Hence, the design of the outer surface of the S. uberis cell apparently provides a “magic hood” preventing recognition of the pathogen by the MEC.
S. uberis activates immune response in macrophages
We were curious to learn if the sluggish immune response towards S. uberis was peculiar to the MEC phenotype and therefore stimulated the murine macrophage model cell RAW264.7 with the very same S. uberis preparations as used before. We validated in a separate study (Günther J, Koy, M, Schuberth, HJ, Seyfert HM; unpublished, manuscript in preparation) that the pathogen-species specific immune response of the RAW264.7 is very similar as recorded from primary bovine monocyte derived macrophages (MDM). There is some evidence that S. uberis may be phagocytosed by macrophages [64, 65], but to the best of our knowledge quantification of the immune response of macrophages against S. uberis has not yet been reported. Using preparations from four different strains we found invariantly that they induced a strong expression of cytokine and chemokine encoding genes. This was very likely caused by strong PRR mediated signaling, since the challenges strongly activated the NF-κB factor complex. These data show very clearly that professional immune cells are capable of recognizing S. uberis as a threatening pathogen. One of the key functions of these cells is to engulf and digest pathogens. Phagocytosis is not a key function of epithelial cells, such as MEC. Hence, it was perhaps not surprising to find that eventually invaded S. uberis pathogens may persist as structurally intact particles inside of MEC . However, intra-cellular digestion of Gram-positive bacteria and subsequent activation of intracellular PRRs, such as TLR9 or NOD2 was found in many cases to be pivotal for mounting an adequate immune response against those invaders (see review ). Hence, data from this study here highlight in turn that the failure of the MEC to respond to the S. uberis challenge is specifically related to the MEC phenotype.
In summary, we demonstrated here that all the strains from our large collection of S. uberis isolates from clinical and subclinical cases of mastitis evaded the immune surveillance of the MEC, representing by far the most abundant first line sentinels of the udder. Failure to activating their immune alert early on after infection explains the commonly observed belated and weak onset of udder inflammation during S. uberis mastitis. We proved, on the other hand that macrophages can indeed mount a vigorous immune response against S. uberis. Hence, our data collectively imply that the observed large fraction of subclinical mastitis associated with S. uberis infections is determined by the pathogen-species specific immune response of MEC. The sometimes occurring severe cases of clinical mastitis after S. uberis infection may relate to specific properties of the individual cow, conceivably including an altered setting and equipment of the udder with resident professional immune cells.
nuclear factor kappa-light-chain-enhancer of activated B-cells
pattern recognition receptor
reverse transcription quantitative PCR
Ericsson Unnerstad H, Lindberg A, Persson Waller K, Ekman T, Artursson K, Nilsson-Öst M, Bengtsson B (2009) Microbial aetiology of acute clinical mastitis and agent-specific risk factors. Vet Microbiol 137:90–97
Zadoks R, Middleton J, McDougall S, Katholm J, Schukken Y (2011) Molecular epidemiology of mastitis pathogens of dairy cattle and comparative relevance to humans. J Mammary Gland Biol Neoplasia 16:357–372
Zadoks RN, Gillespie BE, Barkema HW, Sampimon OC, Oliver SP, Schukken YH (2003) Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds. Epidemiol Infect 130:335–349
Douglas VL, Fenwick SG, Pfeiffer DU, Williamson NB, Holmes CW (2000) Genomic typing of Streptococcus uberis isolates from cases of mastitis, in New Zealand dairy cows, using pulsed-field gel electrophoresis. Vet Microbiol 75:27–41
Hossain M, Egan SA, Coffey T, Ward PN, Wilson R, Leigh JA, Emes RD (2015) Virulence related sequences; insights provided by comparative genomics of Streptococcus uberis of differing virulence. BMC Genomics 16:334
Hill AW (1988) Pathogenicity of two strains of Streptococcus uberis infused into lactating and non-lactating bovine mammary glands. Res Vet Sci 45:400–404
Tassi R, McNeilly TN, Fitzpatrick JL, Fontaine MC, Reddick D, Ramage C, Lutton M, Schukken YH, Zadoks RN (2013) Strain-specific pathogenicity of putative host-adapted and nonadapted strains of Streptococcus uberis in dairy cattle. J Dairy Sci 96:5129–5145
Rato MG, Bexiga R, Nunes SF, Cavaco LM, Vilela CL, Santos-Sanches I (2008) Molecular epidemiology and population structure of bovine Streptococcus uberis. J Dairy Sci 91:4542–4551
Abureema S, Smooker P, Malmo J, Deighton M (2014) Molecular epidemiology of recurrent clinical mastitis due to Streptococcus uberis: evidence of both an environmental source and recurring infection with the same strain. J Dairy Sci 97:285–290
Lundberg A, Nyman A, Unnerstad H, Waller K (2014) Prevalence of bacterial genotypes and outcome of bovine clinical mastitis due to Streptococcus dysgalactiae and Streptococcus uberis. Acta Vet Scand 56:80
Tomita T, Meehan B, Wongkattiya N, Malmo J, Pullinger G, Leigh J, Deighton M (2008) Identification of Streptococcus uberis multilocus sequence types highly associated with mastitis. Appl Environ Microbiol 74:114–124
Bannerman DD, Paape MJ, Goff JP, Kimura K, Lippolis JD, Hope JC (2004) Innate immune response to intramammary infection with Serratia marcescens and Streptococcus uberis. Vet Res 35:681–700
Leigh JA, Egan SA, Ward PN, Field TR, Coffey TJ (2010) Sortase anchored proteins of Streptococcus uberis play major roles in the pathogenesis of bovine mastitis in dairy cattle. Vet Res 41:63
Swanson KM, Stelwagen K, Dobson J, Henderson HV, Davis SR, Farr VC, Singh K (2009) Transcriptome profiling of Streptococcus uberis-induced mastitis reveals fundamental differences between immune gene expression in the mammary gland and in a primary cell culture model. J Dairy Sci 92:117–129
Moyes K, Drackley J, Morin D, Bionaz M, Rodriguez-Zas S, Everts R, Lewin H, Loor J (2009) Gene network and pathway analysis of bovine mammary tissue challenged with Streptococcus uberis reveals induction of cell proliferation and inhibition of PPARgamma signaling as potential mechanism for the negative relationships between immune response and lipid metabolism. BMC Genomics 10:542
de Greeff A, Zadoks R, Ruuls L, Toussaint M, Nguyen TKA, Downing A, Rebel J, Stockhofe-Zurwieden N, Smith H (2013) Early host response in the mammary gland after experimental Streptococcus uberis challenge in heifers. J Dairy Sci 96:3723–3736
Günther J, Koczan D, Yang W, Nürnberg G, Repsilber D, Schuberth HJ, Park Z, Macbool N, Molenaar A, Seyfert H-M (2009) Assessment of the immune capacity of mammary epithelial cells: comparison with mammary tissue after challenge with Escherichia coli. Vet Res 40:31
Mitterhuemer S, Petzl W, Krebs S, Mehne D, Klanner A, Wolf E, Zerbe H, Blum H (2010) Escherichia coli infection induces distinct local and systemic transcriptome responses in the mammary gland. BMC Genomics 11:138
Petzl W, Zerbe H, Günther J, Yang W, Seyfert H-M, Schuberth HJ (2008) Escherichia coli, but not Staphylococcus aureus triggers an early increased expression of factors contributing to the innate immune defense in the udder of the cow. Vet Res 39:18
Leigh JA, Field TR, Williams MR (1990) Two strains of Streptococcus uberis, of differing ability to cause clinical mastitis, differ in their ability to resist some host defence factors. Res Vet Sci 49:85–87
Capuco AV, Akers RM, Smith JJ (1997) Mammary growth in Holstein cows during the dry period: quantification of nucleic acids and histology. J Dairy Sci 80:477–487
Capuco AV, Wood DL, Baldwin R, Mcleod K, Paape MJ (2001) Mammary cell number, proliferation, and apoptosis during a bovine lactation: relation to milk production and effect of bST. J Dairy Sci 84:2177–2187
Rainard P, Riollet C (2006) Innate immunity of the bovine mammary gland. Vet Res 37:369–400
Schukken YH, Günther J, Fitzpatrick J, Fontaine MC, Goetze L, Holst O, Leigh J, Petzl W, Schuberth HJ, Sipka A, Smith DGE, Quesnell R, Watts J, Yancey R, Zerbe H, Gurjar A, Zadoks RN, Seyfert HM (2011) Host-response patterns of intramammary infections in dairy cows. Vet Immunol Immunopathol 144:270–289
Strandberg Y, Gray C, Vuocolo T, Donaldson L, Broadway M, Tellam R (2005) Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells. Cytokine 31:72–86
Goldammer T, Zerbe H, Molenaar A, Schuberth HJ, Brunner RM, Kata SR, Seyfert HM (2004) Mastitis increases mammary mRNA abundance of β-defensin 5, toll-like-receptor 2 (TLR2), and TLR4 but not TLR9 in cattle. Clin Diagn Lab Immunol 11:174–185
Yang W, Molenaar AJ, Kurts-Ebert B, Seyfert HM (2006) NF-κB factors are essential, but not the switch, for pathogen-related induction of the bovine β-defensin 5-encoding gene in mammary epithelial cells. Mol Immunol 43:210–225
Porcherie A, Cunha P, Trotereau A, Roussel P, Gilbert F, Rainard P, Germon P (2012) Repertoire of Escherichia coli agonists sensed by innate immunity receptors of the bovine udder and mammary epithelial cells. Vet Res 43:14
Wellnitz O, Berger U, Schaeren W, Bruckmaier R (2012) Mastitis severity induced by two Streptococcus uberis strains is reflected by the mammary immune response in vitro. Schweiz Arch Tierheilkd 154:317–323
Takeuchi O, Akira S (2010) Pattern recognition receptors and inflammation. Cell 140:805–820
Akira S, Uematsu S, Takeuchi O (2006) Pathogen recognition and innate immunity. Cell 124:783–801
Karin M, Lin A (2002) NF-kappaB at the crossroads of life and death. Nat Immunol 3:221–227
Hoesel B, Schmid J (2013) The complexity of NF-kappaB signaling in inflammation and cancer. Mol Cancer 12:86
Oeckinghaus A, Hayden MS, Ghosh S (2011) Crosstalk in NF-κB signaling pathways. Nat Immunol 12:695–708
Drennan MB, Nicolle D, Quesniaux VJF, Jacobs M, Allie N, Mpagi J, Fremond C, Wagner H, Kirschning C, Ryffel B (2004) Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection. Am J Pathol 164:49–57
Elson G, Dunn-Siegrist I, Daubeuf B, Pugin J (2006) Contribution of Toll-like receptors to the innate immune response to Gram-negative and Gram-positive bacteria. Blood 109:1574–1583
Asplin IR, Carl DJ, Way SS, Jones AL (2008) Role of Toll-like receptor 2 in innate resistance to Group B Streptococcus. Microb Pathog 44:43–51
Jin MS, Kim SE, Heo JY, Lee ME, Kim HM, Paik SG, Lee H, Lee JO (2007) Crystal structure of the TLR1-TLR2 heterodimer induced by binding of a tri-acylated lipopeptide. Cell 130:1071–1082
Uematsu S, Akira S (2008) Toll-Like receptors (TLRs) and their ligands. Handb Exp Pharmacol 2008:1–20
Flo TH, Halaas Ø, Lien E, Ryan L, Teti G, Golenbock DT, Sundan A, Espevik T (2000) Human toll-like receptor 2 mediates monocyte activation by Listeria monocytogenes, but not by group B streptococci or lipopolysaccharide. J Immunol 164:2064–2069
Farhat K, Sauter KS, Brcic M, Frey J, Ulmer AJ, Jungi TW (2008) The response of HEK293 cells transfected with bovine TLR2 to established pathogen-associated molecular patterns and to bacteria causing mastitis in cattle. Vet Immunol Immunopathol 125:326–336
Ward PN, Field TR, Ditcham WGF, Maguin E, Leigh JA (2001) Identification and disruption of two discrete loci encoding hyaluronic acid capsule biosynthesis genes hasA, hasB, and hasC in Streptococcus uberis. Infect Immun 69:392–399
Czabanska A (2013) Immunochemical investigations of the cell envelope components isolated from Streptococcus uberis. PhD Thesis, University of Lübeck, Germany
Peterson GL (1979) Review of the Folin phenol protein quantitation method of Lowry, Rosebrough, Farr and Randall. Anal Biochem 100:201–220
Morath S, Geyer A, Hartung T (2001) Structure-function relationship of cytokine induction by lipoteichoic acid from Staphylococcus aureus. J Exp Med 193:393–398
Czabanska A, Neiwert O, Lindner B, Leigh J, Holst O, Duda KA (2012) Structural analysis of the lipoteichoic acids isolated from bovine mastitis Streptococcus uberis 233, Streptococcus dysgalactiae 2023 and Streptococcus agalactiae 0250. Carbohyd Res 361:200–205
Seo HS, Nahm MH (2009) Lipoprotein lipase and hydrofluoric acid deactivate both bacterial lipoproteins and lipoteichoic acids, but platelet-activating factor-acetylhydrolase degrades only lipoteichoic acids. Clin Vaccine Immunol 16:1187–1195
Bauer I, Günther J, Wheeler TT, Engelmann S, Seyfert H-M (2015) Extracellular milieu grossly alters pathogen-specific immune response of mammary epithelial cells. BMC Vet Res 11:67
Yang W, Zerbe H, Petzl W, Brunner RM, Günther J, Draing C, von Aulock S, Schuberth HJ, Seyfert HM (2008) Bovine TLR2 and TLR4 properly transduce signals from Staphylococcus aureus and E. coli, but S. aureus fails to both activate NF-κ B in mammary epithelial cells and to quickly induce TNFα and interleukin-8 (CXCL8) expression in the udder. Mol Immunol 45:1385–1397
Ito S, Chen C, Satoh J, Yim S, Gonzalez FJ (2007) Dietary phytochemicals regulate whole-body CYP1A1 expression through an arylhydrocarbon receptor nuclear translocator-dependent system in gut. J Clin Invest 117:1940–1950
Fujii-Kuriyama Y, Kawajiri K (2010) Molecular mechanisms of the physiological functions of the aryl hydrocarbon (dioxin) receptor, a multifunctional regulator that senses and responds to environmental stimuli. Proc Jpn Acad Ser B Phys Biol Sci 86:40–53
Badal S, Delgoda R (2014) Role of the modulation of CYP1A1 expression and activity in chemoprevention. J Appl Toxicol 34:743–753
Field TR, Ward PN, Pedersen LH, Leigh JA (2003) The hyaluronic acid capsule of streptococcus uberis is not required for the development of infection and clinical mastitis. Infect Immun 71:132–139
Günther J, Esch K, Poschadel N, Petzl W, Zerbe H, Mitterhuemer S, Blum H, Seyfert HM (2011) Comparative kinetics of Escherichia coli- and Staphylococcus aureus-specific activation of key immune pathways in mammary epithelial cells demonstrates that S. aureus elicits a delayed response dominated by interleukin-6 (IL-6) but not by IL-1A or tumor necrosis factor alpha. Infect Immun 79:695–707
Okumura CY, Nizet V (2014) Subterfuge and sabotage: evasion of host innate defenses by invasive gram-positive bacterial pathogens. Annu Rev Microbiol 68:439–458
Foster TJ (2009) Colonization and infection of the human host by staphylococci: adhesion, survival and immune evasion. Vet Dermatol 20:456–470
Moschioni M, Pansegrau W, Barocchi MA (2010) Adhesion determinants of the Streptococcus species. Microb Biotechnol 3:370–388
Sander LE, Davis MJ, Boekschoten MV, Amsen D, Dascher CC, Ryffel B, Swanson JA, Muller M, Blander JM (2011) Detection of prokaryotic mRNA signifies microbial viability and promotes immunity. Nature 474:385–389
Fedtke I, Mader D, Kohler T, Moll H, Nicholson G, Biswas R, Henseler K, Götz F, Zähringer U, Peschel A (2007) A Staphylococcus aureus ypfP mutant with strongly reduced lipoteichoic acid (LTA) content: LTA governs bacterial surface properties and autolysin activity. Mol Microbiol 65:1078–1091
Ray A, Cot M, Puzo G, Gilleron M, Nigou J (2013) Bacterial cell wall macroamphiphiles: pathogen-/microbe-associated molecular patterns detected by mammalian innate immune system. Biochimie 95:33–42
Henneke P, Morath S, Uematsu S, Weichert S, Pfitzenmaier M, Takeuchi O, Müller A, Poyart C, Akira S, Berner R, Teti G, Geyer A, Hartung T, Trieu-Cuot P, Kasper DL, Golenbock DT (2005) Role of lipoteichoic acid in the phagocyte response to group B Streptococcus. J Immunol 174:6449–6455
Schröder NWJ, Morath S, Alexander C, Hamann L, Hartung T, Zähringer U, Göbel UB, Weber JR, Schumann RR (2003) Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide-binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved. J Biol Chem 278:15587–15594
Schmidt RR, Pedersen CM, Qiao Y, Zähringer U (2011) Chemical synthesis of bacterial lipoteichoic acids: an insight on its biological significance. Org Biomol Chem 9:2040–2052
Grant RG, Finch JM (1997) Phagocytosis of Streptococcus uberis by bovine mammary gland macrophages. Res Vet Sci 62:74–78
Almeida RA, Oliver SP (1995) Phagocytosis of Streptococcus uberis by bovine mammary macrophages: opsonizing effect of bovine antiserum. Zentralbl Veterinarmed B 42:331–337
Matthews KR, Almeida RA, Oliver SP (1994) Bovine mammary epithelial cell invasion by Streptococcus uberis. Infect Immun 62:5641–5646
Ki Shibata, Hasebe A, Sasaki T, Watanabe T (1997) Mycoplasma salivarium induces interleukin-6 and interleukin-8 in human gingival fibroblasts. FEMS Immunol Med Microbiol 19:275–283
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917
AC conducted the biochemical isolations of the cell envelop components and established the 0140J::ISS1 P’ mutant; IB conducted the challenges with live bacteria; JG conducted most of the experiments, analyzed the data and drafted the manuscript; JAL helped constructing that mutant and provided advice regarding experimental design and data interpretation. He, OH and HMS had conceived the study and contributed interpreting the data and finalizing the manuscript. All authors read and approved the final manuscript.
We are grateful for the expert technical assistance by Angelika Deike, Bärbel Pletz and Emma Schröder. This study was supported by Pfizer/Zoetis through the Mastitis Consortium. We are also grateful to Dr. Michael Fontaine (Moredun Research Instutute, UK) for providing additional S. uberis strains.
The authors declare that they have no competing interests.
About this article
Cite this article
Günther, J., Czabanska, A., Bauer, I. et al. Streptococcus uberis strains isolated from the bovine mammary gland evade immune recognition by mammary epithelial cells, but not of macrophages. Vet Res 47, 13 (2016). https://doi.org/10.1186/s13567-015-0287-8
- Mammary Epithelial Cell
- Immune Gene
- Clinical Mastitis
- Subclinical Mastitis