Exploring the Staphylococcus aureus cell wall for invariant immunodominant targets
Mora Hernández, Yaremit
DOI:
10.33612/diss.147005930
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Publication date: 2020
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Mora Hernández, Y. (2020). Exploring the Staphylococcus aureus cell wall for invariant immunodominant targets. University of Groningen. https://doi.org/10.33612/diss.147005930
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Chapter 5
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Ever since the domestication of animals to produce food for human consumption, livestock farmers and consumers have been at risk of contracting zoonotic diseases, either through direct contacts with the animals or consumption of contaminated food products. Since the middle of the last century, the risks of bacterial infections amongst livestock and the spread of zoonotic bacterial diseases to humans have been mitigated through the preventive or therapeutic administration of antibiotics. Unfortunately, however, these powerful drugs are becoming increasingly less effective due to the widespread emergence of antimicrobial resistance as a consequence of the overuse, wrong use and release into the environment of antibiotics. To stop this worrisome trend, so-called ‘one health’ approaches will be needed that take into account the close connection between the health of people and animals as well as our shared environment (https://www.cdc.gov/onehealth/).
A clear example of a veterinary disease that calls for a one health approach is bovine mastitis. This intramammary gland infection causes significant economic losses due to a combination of reduced milk production, discarded milk, lowered milk product quality, costs for veterinary services and culling. Moreover, it affects the welfare of the cattle and may lead to the emergence of pathogens with zoonotic potential. The latter view is underscored by the fact that one of the main causative agents of mastitis is the Gram-positive bacterium Staphylococcus aureus. This notoriously drug resistant pathogen causes infections in humans and livestock, and it is often present in bovine mastitic milk
1.
S. aureus is a frequent component of the microbiota of humans and animals, which carry this bacterium most of the time asymptomatically. However, when given the chance through trauma, ineffective skin or mucosal barriers, or a weakened immune system, S. aureus can cause a wide range of potentially deadly diseases. The transmission of S. aureus is difficult to control due to its omnipresence and the ease at which it colonizes the mammalian skin and mucosa. Also, this pathogen makes use of numerous different virulence factors that allow it to colonize host cells and tissues, to invade the host, and to evade the host’s immune defenses 2. To make matters worse, S. aureus rapidly
develops resistance to antibiotics, either through adaptive responses, through mutations, or through the acquisition of resistance genes by horizontal gene transfer. The latter is painfully underscored by the emergence of
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resistant S. aureus (MRSA) lineages in humans and livestock, which hasbecome a serious global health concern. Also in dairy farming, antimicrobial resistant S. aureus has become a significant problem, especially since the first approach to treat and control mastitis is the administration of antibiotics 3. For
this reason, additional means to prevent and treat mastitis are urgently needed. In particular, immunization approaches can provide effective protection against infections. However, despite various attempts, no vaccine that protects against S. aureus infections has thus far been brought to the market 4,5. The vaccination
approaches that have been tried involved immunization with inactivated bacteria, inactive toxins or conserved virulence factors, but they did not lead to effective prevention, control or treatment of bovine mastitis caused by S. aureus.
A general approach that may be followed to develop vaccines against staphylococcal infections in general, and bovine mastitis in particular, could involve the following steps: i. isolation and characterization of representative clinical isolates; ii. screening for invariable components of collected clinical isolates, especially virulence factors on the bacterial cell surface, that are readily recognized by the immune system of the mammalian host; iii. selection and production of the potential antigens to be used; iv. testing whether the selected antigens elicit protective immunity in relevant animal infection models; v. analysis of the elucidated immune responses; vi. safety testing and toxicology; and vii clinical trials. All steps in this process are challenging and require careful consideration. However, one of the prime hurdles in the early stages of the process concerns the identification of the right antigens, which is particularly relevant in view of the many different S. aureus lineages, their high genomic plasticity, and the even higher variability in the presentation of potential antigens on the staphylococcal cell surface6,7. Moreover, in recent years it has
become increasingly clear that potentially attractive proteinaceous vaccine targets on the S. aureus cell surface may present multiple immunodominant epitopes, but that not all immunoglobulins binding to these epitopes will protect the host against infection. It thus seems important to pinpoint and characterize those domains that are most likely to elicit protective immune responses upon immunization 8–10.
The present PhD thesis describes the steps that have been followed to explore the S. aureus cell wall for invariant immunodominant targets that could be
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applied in future vaccines for the prevention of staphylococcal infections in general, and bovine mastitis in particular. The respective steps that were undertaken are graphically represented in Figure 1.
Figure 1. Pipeline for the identification of immunodominant vaccine targets and potentially protective immune responses. Identification and typing of S. aureus isolates from milk samples obtained from cows suffering from mastitis was performed by MLVF and spa-typing. Identification of surface-exposed or cell wall-located proteins from representative S. aureus isolates was achieved by proteomics, using cell surface shaving with trypsin and cell wall protein extraction with KSCN. Cell surface antigens were produced and isolated using a Lactococcus lactis-based expression system. The purified recombinant antigens were individually used for immunization of mice and potentially protective immune responses were assessed after infection with S. aureus in a bacteremia model. Finally, several antigen domains were used to map the binding of IgGs from immunized mice, colonized humans and cows suffering from mastitis.
Chapter 2 of this thesis describes the collection of 33 S. aureus isolates from mastitic milk obtained from dairy farms in the Comarca Lagunera region in Mexico. The isolates were characterized and typed to determine the respective S. aureus linages causing the bovine mastitis. Using multi-locus variable number tandem repeat (VNTR) fingerprinting (MLVF), the 33 isolates were
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classified in four different groups. Subsequent spa-typing showed that two ofthese MLVF groups were composed of isolates with the spa-type t224, while another group contained isolates with the spa-types t416 and t3196, and the fourth MLVF group contained isolates with the spa-type t114. MLVF is a typing method that is quick, cheap, highly discriminatory and complementary to other typing methods, such as spa-typing. The main downside of MLVF is that it is difficult to compare results between laboratories. On the other hand, spa-typing is somewhat less discriminatory, because it is based on variations in a single locus, but its DNA sequence-based output allows comparisons between labs. Following the spa-typing, six isolates were selected from the distinguished mastitis isolate groups for further analyses. These included three isolates with the spa-type t224, because most of the collected isolates had this spa-type. To further characterize the six selected isolates genetically, multi-locus sequence typing (MLST) was applied. Originally, this approach involved the PCR amplification and subsequent sequencing of seven house-keeping genes, allowing the designation of a sequence type (ST)11. The main advantages of this
method are the unambiguity and high reproducibility of the sequencing-based results 12. However, as the costs for whole-genome sequencing have gradually
declined, MLST of the six selected mastitis isolates was performed based on their whole genome sequences. This resulted in the identification of two new ST types, namely ST4551 and ST4552. The isolate G-1 belongs to ST4551, while the other five selected isolates all belong to ST4552. Subsequent studies on isolate G-1, described in Chapter 3, showed that this isolate is indeed different from the other isolates with respect to the predicted numbers of extracytoplasmic proteins at different subcellular locations as well as its protein expression.
To pinpoint potential proteinaceous vaccine targets, proteomics approaches are of great value. In most of the previous proteome studies on mastitis isolates, which were aimed at identifying virulence factors or antigens, the strains were grown on rich media in order to obtain sufficient amounts of cellular or extracellular proteins. For instance, in a study performed in 2007, the cell wall-associated proteins of a S. aureus isolate implicated in bovine mastitis were characterized after growth of the bacteria on blood agar plates 13. The same
condition was applied in a subsequent immunoproteomics analysis on S. aureus isolates causing sub-clinical mastitis 14. In another previous study, several S.
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aureus isolates from mastitis cases were grown in tryptic soy broth prior to the proteome analysis 15. Furthermore, in an attempt to identify multiple antigens
presented on the surface of S. aureus cells, the expression of virulence-related surface- or cell wall-anchored proteins upon growth in a low-iron medium was explored 16. Yet, the most realistic view on the proteome composition of a
pathogen will be obtained by using a growth medium that closely mimics the conditions in the host. For example, to mimic the conditions of bacteremia, S. aureus was previously exposed to human plasma 17. This actually appeared to
impose conditional constraints on the bacteria that were closely mimicked by the Roswell Park Memorial Institute (RPMI) 1640 medium, as judged by genome-wide transcript profiles 18. Accordingly, to mimic the conditions in the
cistern of the bovine mammary gland for the present studies, whey permeate was applied to grow staphylococcal mastitis isolates. This growth medium is predominantly composed of milk proteins and lactose. In fact, whey permeate has been used most widely to grow and maintain starter cultures of lactic acid bacteria. Although growth in whey permeate was slower than in richer growth media, sufficient proteinaceous material could be obtained, both for Western blotting and proteome analyses. Indeed, as documented in Chapter 3, differences in protein expression were observed when the mastitis isolates were grown in whey permeate or RPMI. Interestingly, this analysis also showed that the abundance of the milk proteins in the supernatants of the whey permeate cultures was very high compared to the proteins expressed and secreted by the S. aureus isolates. However, because the present studies were aimed at identifying the cell wall-localized and cell surface-exposed staphylococcal proteins (the surfacome), the presence of the milk proteins in the medium did not limit the detection of these staphylococcal proteins. Since these exposed proteins represent the first mediators of interactions between the pathogen and its host, they are the most prominent candidates for antigen selection.
To profile the cell surface proteins of the six Mexican S. aureus isolates associated with bovine mastitis, two different sampling approaches were employed, namely: i. shaving of the bacterial surface with immobilized trypsin, which allows identification of exposed protein domains; and ii. the extraction of cell wall-associated proteins using the chaotropic agent potassium thiocyanate (KSCN) (Chapter 3). The proteins extracted by the KSCN treatment are associated non-covalently to the cell wall by hydrogen bonds, van der Waals forces, or hydrophobicity. Conversely, the shaving approach allowed also the
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identification of surface-exposed LPXTG proteins that are covalently bound tothe peptidoglycan by sortases. Although the bacterial cells harvested after growth in whey permeate were stringently washed, various milk proteins were still detected by both approaches. This implies that the bacteria had acquired a ‘corona’ of milk proteins that are tightly bound to the bacterial cell surface. The identification of this corona is important, as it may influence the bacterial properties substantially. For instance, it can influence the bacterial interactions with host tissues, and make S. aureus invisible for the bovine immune system as the bacteria will be covered by proteins of the host. More specifically the milk proteins may shield potential vaccine targets against binding of immunoglobulins, thereby leading to immune evasion.
The types of staphylococcal proteins that can be identified by the different approaches to profile the staphylococcal cell wall proteome are quite heterogeneous, including not only the proteins with typical domains for covalent or non-covalent cell wall attachment, but also many cytoplasmic and membrane proteins. In a study performed by Solis et al., the shaving of S. aureus cells resulted in the identification of so-called moonlighting cytoplasmic proteins. The presence of these proteins was explained by the possible export via unknown secretion mechanisms, re-association to the cell-wall following autolysis or diffusion through holins 19. Other explanations for the detection of cytoplasmic
proteins upon cell surface shaving could be membrane weakening by cytolytic toxins, autolysis or lysis related to prophage activity 6,20,21. Indeed, S. aureus
cells produce a range of differentially expressed peptidoglycan hydrolases and they usually harbor several prophages in their genome that can be induced by stressful DNA-damaging conditions. This may also be an explanation for the variations in the cytoplasmic proteins that were associated with the six investigated mastitis isolates as described in Chapter 3. In fact, several phage proteins were detected by the proteome analyses of the five ST4552 isolates, while for the G-1 isolate with ST4551 no phage proteins were identified. The latter strain displayed also the lowest number of cytoplasmic proteins on its cell surface, as inferred from the cell wall extraction with KSCN and shaving with trypsin. This is a clear indication for the involvement of phages in the release of cytoplasmic proteins with possible moonlighting functions by the five ST4552 isolates.
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Comparison of the protein identifications upon cell wall shaving and KSCN treatment only revealed an overlap of 28,29%, which shows that these analyses are complementary. For example, the proteins LytM and SceD were only identified in the surface shaving analysis, indicating that these proteins are located at exposed positions in the S. aureus cell wall. In contrast the Sle1 protein was only detected upon cell-wall extraction with KSCN, suggesting it is located at a deeper position in the cell wall. However, the absence of trypsin cleavage of Sle1 could also relate to shielding by milk proteins as suggested above, or to a tight three-dimensional protein structure that protects against trypsin cleavage.
Among the proteins identified by proteomics, multiple peptidoglycan hydrolases were detected, which raised the question whether such proteins could be potential vaccine targets. The peptidoglycan hydrolases Aly, LytM and Sle1 were therefore selected for further analyses, which involved their production in the heterologous expression host Lactococcus lactis and subsequent purification (Chapter 4). Importantly, not only the full-size proteins, but also particular domains of Aly, LytM and Sle1 were produced and purified. To determine whether the isolated proteins and domains would assume their natural conformation, their biological activity was determined. Indeed, enzymatic activity was shown for all of the active site domains.
Immunization of mice with either of the three full-size peptidoglycan hydrolases resulted in high IgG responses. However, the high IgG levels did unfortunately not result in a protection of the mice against death by S. aureus bacteremia. Of note, the respective IgG levels against Aly, LytM and Sle1 were comparable to what was observed in an earlier study, where mice were immunized with an octavalent cocktail of the S. aureus IsaA, LytM, Nuc, pro-Atl proteins plus four phenol-soluble modulins α 22. After three subcutaneous injections, high IgG
responses against all antigens were detected. However, as subsequently shown in S. aureus bacteremia and skin infection models, upon vaccination with this antigen cocktail, no reduction in the S. aureus load was observed in the bacteremia model as compared to the control. Also, in the skin infection model, no differences were detectable with respect to discomfort scores and animal survival rates between mice immunized with the cocktail of S. aureus antigens and placebo-immunized mice 22.
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To shed more light on the possible reasons why immunization with the separateAly, LytM or Sle1 antigens failed in the present studies to protect mice against S. aureus infection, the specificity of the respective IgGs for various domains of these three antigens was determined. As most of the peptidoglycan hydrolases are composed of a cell wall binding domain, an active site domain and, in some cases, an activity-controlling domain, the recognition of each individual domain by the murine IgGs was individually inspected as described in Chapter 4. To this end, the separate domains, as recombinantly expressed in L. lactis and subsequently purified, were employed in a Western blotting analysis. In addition to binding of the purified domains by IgGs from immunized mice, the binding by IgGs from healthy human volunteers and from heavily S. aureus-colonized patients with the genetic blistering disease epidermolysis bullosa (EB) was inspected. The results presented in Chapter 4 show that the three peptidoglycan hydrolases Aly, LytM and Sle1 elicited strong IgG responses in humans and in mice. However, the particular domains that were bound differed substantially. In particular, the serum IgGs of immunized mice were shown to target the LysM cell wall-binding domain of Sle1 and the catalytic domains of Aly and LytM. On the other hand, the human sera were shown to contain IgGs that target the catalytic domain of Sle1 and the N-terminal domains of Aly and LytM. This makes the latter three domains interesting for future immunization studies, especially since patients with EB were previously shown to mount elevated IgG responses against S. aureus that are potentially protective against severe staphylococcal infections 23. In this respect it is noteworthy that in a
previous study, epitope mapping showed that a conserved 62-residue N-terminal domain of IsaA was recognized by IgGs from EB patient sera, but not by IgGs from mice immunized with IsaA. Also in this case, the murine IgGs were non-protective and exhibited a high binding preference towards the C-terminal domain of IsaA. This observation suggested that IgG responses against cell surface-exposed S. aureus proteins may be host specific. However, in this respect it should be taken into account how a surface protein is presented to the host, i.e. as a purified protein as was done in the murine immunizations, or exposed on the surface of colonizing or infecting S. aureus cells. Judged by the previous studies on IsaA, such differences may be crucial, because monoclonal antibodies against the N-terminal domain of IsaA were shown to confer a certain level of protection against S. aureus infections in different murine models 8,10,24– 26. This suggests that it will be beneficial for vaccine production to focus
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attention on those domains that elicited potentially protective IgG responses during (long-term) colonization of the host.
In the context of the present PhD research, the investigations on murine and human IgG responses towards different domains of surface-exposed S. aureus proteins raised the question whether cows suffering from mastitis would also mount preferential IgG responses to particular domains of proteins presented on the surface of S. aureus isolates associated with mastitis. In particular, the surface proteome analyses of the six Mexican mastitis-associated S. aureus isolates had shown surface exposure of the cell wall hydrolases IsaA, Sle1 and Aly (Chapter 3). Therefore, in a pilot study, sera from cows suffering from mastitis were used to assess possible IgG responses against different purified domains of IsaA, Sle1 and Aly that had been recombinantly expressed in L. lactis. For the cows of which the sera were obtained, the causative S. aureus strains were isolated and the presence of the isaA, sle1 and aly genes was verified by sequencing, showing that they encoded proteins with nearly 100% sequence identity to the respective proteins of the six S. aureus mastitis-associated isolates described in Chapters 3 and 4. As shown in Figure 2, unlike the human sera, the sera from nearly all cows with mastitis lacked IgGs against IsaA and its subdomains. Only the cow serum sample 1280 contained IgGs that bind to the full-size IsaA and its separately expressed N- and C-terminal domains. Nonetheless, Western blotting using cell and growth medium fractions of the mastitis isolates grown in whey permeate did reveal IsaA production and secretion by the bacteria (Figure 3). This suggests the existence of species-specific differences in the elicited IgG responses, or differences in the presentation of IsaA to the bovine, murine and human immune systems. Furthermore, all cow sera contained IgGs against the LysM domain of Sle1, while no IgGs against the active site CHAP domain were detected (Figure 2; see Chapter 4 for a schematic representation of the Sle1 domains). This specific IgG response with preferential recognition of the LysM domain is similar to the one observed for sera from mice immunized with Sle1. In contrast, several investigated human sera did contain IgGs against the CHAP domain as described in Chapter 4 of this thesis. This provides additional evidence for a species-specific preferential domain recognition by the immune systems of cows, mice and humans.
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Figure 2: Specific binding of IgGs from mastitic cow sera to IsaA and Sle1.The full-size IsaA, protein and its N- and C-terminal domains (IsaA-N, IsaA-C), as well as the full-full-size Sle1 protein and its LysM and CHAP domains were recombinantly expressed in L. lactis with a C-terminal His6-tag. Upon metal-affinity purification, the different proteins were separated by LDS-PAGE and transferred to nitrocellulose membranes for immunodetection of specific IgGs in sera from twelve cows with mastitis caused by S. aureus. The indicated numbers of the cow sera correspond to the respective S. aureus isolate numbers. The presence of the recombinantly produced proteins was visualized by SimplyBlue Safe-staining and immunoblotting with Anti-His antibodies. Binding of bovine IgGs was visualized with IRDye CW800-labelled goat anti-bovine secondary antibodies. For a description of the antigen expression and purification, and the Western blotting procedure, see Chapters 3 and 4 of this thesis.
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Figure 3: Expression of IsaA and Sle1 by eleven S. aureus isolates from cows with mastitis. S. aureus isolates from cows with mastitis were grown in whey permeate and culture samples were collected in the late exponential growth phase. Subsequently, cells were separated from the growth medium and both fractions were used for LDS-PAGE and Western blotting. The numbers on top of each lane refer to the different investigated S. aureus isolates. (A)Immunodetection of the IsaA protein using the monoclonal antibody 1D9. Purified IsaA was loaded as a control. (B) Immunodetection of Sle1 using the polyclonal anti-AAA rabbit antibody and an IRDye 800CW-labelled goat anti-rabbit secondary antibody. Purified Sle1 was loaded as a control. Molecular weights of marker proteins are indicated on the left of the blots. For a description of the culture conditions and Western blotting procedure, see Chapter 3 of this thesis.
Four sera from cows with mastitis were also used to examine IgG responses against Aly and its three subdomains, referred to as the N-terminal coiled-coil, glucosaminidase (GM) and CHAP domains (see Chapter 4 for a schematic representation of the domains). Nearly identical results were obtained for the four sera tested, showing that the coiled-coil domain was preferentially recognized by IgGs in all four sera (Figure 4). Only the serum sample 1824 contained IgGs that bound to the GM domain. This is again different from the situation observed in humans and immunized mice, where IgG responses to the GM and CHAP domains were quite prominent, while the N-terminal coiled-coil domain was only recognized by human IgGs, albeit as a minor fraction.
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Figure 4. Specific binding of IgGs from mastitic cow sera to Aly. The full-size Aly protein and its three domains (coiled-coil, glucosaminidase and CHAP) were recombinantly expressed in L. lactis with a C-terminal His6-tag. Upon metal-affinity purification, the different proteins were separated by LDS-PAGE and transferred to nitrocellulose membranes for immunodetection of specific IgGs in sera from four cows with mastitis caused by S. aureus. The indicated numbers of the cow sera correspond to the respective S. aureus isolate numbers. The presence of the recombinantly produced proteins was visualized by SimplyBlue Safe-staining and immunoblotting with Anti-His antibodies. Binding of bovine IgGs was visualized with IRDye CW800-labelled goat anti-bovine secondary antibodies. For a description of the antigen expression and purification, and the Western blotting procedure, see Chapters 3 and 4 of this thesis.
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Conclusion
Altogether, the present PhD thesis describes a blueprint approach to identify and characterize potential targets for the development of vaccines against the important human and livestock pathogen S. aureus. The results highlight differential IgG responses of immunized mice, colonized humans, and cows with mastitis to particular staphylococcal antigens. This points out interspecies variations that need to be taken into account when testing candidate vaccine targets of S. aureus in animal models. The studies presented in this thesis indicate that cell wall hydrolases of S. aureus are potentially good candidate targets to elicit strong antibody responses, but it will be a major challenge for future studies to figure out which of the immune responses will indeed be protective against S. aureus infections. As exemplified by previous studies on potentially protective antibody responses against the S. aureus IsaA protein, the best possible way to achieve the ambitious objective of developing an effective anti-S. aureus vaccine will probably involve the generation of monoclonal antibodies that target particular domains of the investigated proteins followed by initial passive immunization studies in mice. This will show whether an IgG response against the respective epitope can be protective. However, in the end, the real proof of efficacy will have to come from active immunization studies with recombinantly produced domains of the most promising antigens in the most relevant hosts, namely humans or cows. The research presented in this PhD thesis could guide the different steps that need to be taken towards the development of the dearly needed vaccines that protect frail individuals from life-threatening staphylococcal diseases, and dairy farmers from the painful losses caused by bovine mastitis.
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