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 1
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Mastitis is the most common livestock disease affecting the dairy industry. It refers to an infection of the mammary gland or udder, which can occur during a period of lactation. Although common in cattle, also other mammals and even humans may be affected by this disease 1. There are different kinds of mastitis, which can be classified in subgroups depending upon different criteria, in particular the lactation stage (non-lactational or lactational mastitis), the course of infection (subacute, acute, per-acute, chronic or recurrent mastitis), or (sub)clinical symptoms 1. During clinical mastitis, a cow shows symptoms like swelling and redness of the mammary gland, increased body temperature and the presence of small clumps in the milk. However, when a cow develops subclinical mastitis, almost no symptoms are observed. A case of subclinical mastitis can, however, be recognized by measuring elevated levels of somatic cells in the milk (higher than 200,000 cells/mL) 2. As a consequence of mastitis, dairy cows show a decreased milk production. The milk losses, together with other costs for treatments, prevention methods and the early culling of cows from the herd result in a large economic loss for the dairy industry 3. The average loss of revenues due to mastitis is estimated to amount €240 per lactating cow per year 4.
Mastitis can be caused by viruses, algae, yeasts and various bacteria, such as coagulase-negative staphylococci (CNS), Staphylococcus aureus, Escherichia coli, Streptococcus uberis and Pseudomonas spp. 5. Worldwide, staphylococci (S. aureus and CNS) are the most frequently encountered causative agents of mastitis1.
Since 1994, it is generally recognized that the pathogen S. aureus is often responsible for causing infections of the mammary gland 6. S. aureus is a Gram-positive, facultative anaerobic bacterium that is part of the microbiota of humans and different animals. This bacterium not only causes mastitis, but also a variety of other infections such as pyoderma, sinusitis and otitis, septicemia, pneumonia, osteomyelitis and prosthetic device infections 7. To colonize its host and cause infections S. aureus produces many different virulence factors, such as surface components that promote adhesion to host cells and tissues, factors that allow evasion of the host’s immune defenses, and toxins that disrupt primary barriers and kill phagocytes 6,8,9. Collectively, these virulence factors are molecules produced by bacteria to occupy different niches in the host that allow the bacteria to grow and reproduce 10. Accordingly, there is a large
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diversity of virulence factors among S. aureus strains that can explain theirpredilection for different types of infections, as exemplified by superantigens that cause the toxic shock syndrome or blistering of the skin in humans 11–13. However, more recently it has become clear that also different metabolic niche adaptations of S. aureus can steer the cause of infection 14,15. On top of this, the status of the host’s immune system, nutrition and genetic predisposition play important roles in the severity of staphylococcal infections 16.
Immune defenses of the mammary gland
During the period of lactation, the mammary gland employs several protective mechanisms against bacterial infections 17. The first line of defense is represented by anatomical barriers, like the teat end (sphincter) and the keratin lining of the teat canals. When this keratin layer becomes damaged, the teat canals show an increased susceptibility for infection 18.
Once the first barrier is overcome, the second line of defense against bacterial infection is formed by immune cells, such as neutrophils, macrophages, and lymphocytes 17. Neutrophils are the principal phagocytic cells in the mammary gland and its secretions during the early stages of infection 19. Their count is low in healthy mammary glands, but represent about 90% of the leukocyte population during mastitis. The main role of neutrophils is to respond to inflammatory mediators for phagocytosis and to kill pathogens non-specifically, for instance by producing small antibacterial peptides and defensins 20. Macrophages form the principal phagocytes in healthy mammary tissues, milk and lactating mammary glands. Like neutrophils, macrophages will phagocytose bacteria non-specifically and destroy them with the help of reactive oxygen species and proteases. However, macrophages facilitate both innate and adaptive immune responses 17. Another important role performed by macrophages is the production of chemotaxis molecules (prostaglandins, leukotrienes, and cytokines) to facilitate neutrophil migration and phagocytic activity 21. Macrophages further support specific immune responses through antigen processing and presentation by the major histocompatibility complex (MHC) class II 22. Lastly, lymphocytes play important roles in both innate and adaptive immunity. These cells recognize antigens through specific receptors, defining the immunological characteristics of diversity, specificity, (non)self-recognition and memory. There are three major kinds of lymphocytes, namely
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T cells, B cells, and natural killer cells. The T cells can be divided in αβ T lymphocytes, including the CD4+ (‘helper cells) and CD8+ (cytotoxic and suppressor, ‘killer cells’) and γδ T lymphocytes. The most abundant T cells in the healthy mammary gland of cows have the CD8+ phenotype, although during mastitis the CD4+ T cells prevail. It has been reported that if, during mastitis caused by S. aureus, activated CD8+ lymphocytes suppress the immune response, the host will be predisposed to develop chronic mastitis 23. Further, it was observed that the presence of subsets of T lymphocytes is depending on the tissue location and the period of lactation 24, whereas this is not the case for B cells 19.
Milk also contains a number of antimicrobial factors, such as the lingual antimicrobial peptides (LAP), lactoferrins and immunoglobulin A (IgA) type antibodies 25. These antimicrobial factors are part of the innate immune system and are always present in the milk, but in varying amounts. Their main function is to provide immediate protection against bacteria entering the mammary gland 26.
A very important contributor to immunity is the complement system, which involves a collection of proteins produced mainly by the liver, monocytes and tissue macrophages. However, the critical complement component C3 is also locally produced in the mammary gland 27. In 2008 it was shown that an intramammary challenge with S. aureus and E. coli increased the expression of the C3 mRNA in mammary epithelial cells 28. The complement system is intimately involved with the innate immune system in control and initiation of inflammation, opsonization of bacterial cell surfaces, attraction and recruitment of phagocytes (involving the C3 and C5 fragments), recognition and ingestion of microorganisms by phagocytosis (involving C3 and C4) and killing of microorganisms 27–30. The complement system is, however, not only part of the innate immune system, but also serves as a bridge between the innate and acquired immune systems 31. In the healthy bovine mammary gland, the lowest concentration of complement proteins is observed during lactation, whereas higher levels are present in the colostrum, the late lactation period and mammary secretions during involution of the mammary gland24.
Cytokines are small secreted-proteins that have important roles in cell signaling, and are produced during inflammatory processes. Because of the high affinity
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of cytokines for their receptors, they can lead to responses at minimalconcentrations 24. Several cytokines have been detected in healthy udders like Interferon (IFN)-γ, Tumor necrosis factor (TNF)-α, Interleukin (IL)-8, and IL-12. During intramammary infection, specific cytokines are down- or up-regulated, depending on the pathogen causing the infection 24. In this context, cytokines have several functions. For example, IFN-γ, besides representing a bridge between the innate and adaptative responses, mediates activation and microbicidal activity of macrophages and neutrophils, induces production of IL-12, and reverses the suppressive effects of the mammary gland secretions24. IL-8 is a pro-inflammatory cytokine and chemoattractant for neutrophils in the mammary gland, induces degranulation and enhances the microbicidal activity of neutrophils 24. IL-1β is another pro-inflammatory cytokine, reported to promote T cell survival and B cell proliferation 32. Furthermore, the pro-inflammatory cytokine IL-17A has been reported to increase the expression of genes encoding chemoattractants, cytokines, and antibacterial proteins33. Lastly, IL-12 also plays a role as a bridge between the innate and adaptative immune systems, stimulating the production of IFN-γ, TNF-α, IL-8 and IL-10, and promoting the production of immunoglobulins involved in pathogen opsonization 24.
Pathology of mastitis
Once S. aureus has entered the mammary gland, it can either assume an extracellular lifestyle by forming biofilms, or an intracellular lifestyle within epithelial cells of the alveoli (Figure 1) 34,35. When a biofilm is formed, bacteria attach to the tissue surface, aggregate and produce a slimy matrix that eventually results in the formation of a mature biofilm, which can subsequently release bacteria again that will spread the infection 36. A study performed in 2007 showed that the ability of S. aureus to produce biofilms increases over time during mastitis 34. In another study, it was shown that the formation of biofilms leads to a decreased susceptibility to antimicrobial agents, which is in fact a general feature of staphylococcal biofilms 37,38. As a consequence, higher concentrations of antimicrobial agents are needed to effectively eliminate the biofilm-embedded bacteria as compared to planktonic bacteria 39.
It has been demonstrated that S. aureus can also enter the epithelial cells, which happens preferentially when the epithelial barrier is damaged 14,40,41. In a
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recent study it was shown that increased levels of transforming growth factor β1 (TGF-β1) in the mammary gland led to increased adhesion and invasion of epithelial cells by S. aureus42. The increased levels of TGF-β1 were thought to increase the expression of fibronectin (Fn) and integrin-β1 (ITGβ1) in the host cells, which are specifically recognized and bound by staphylococcal adhesion factors. During an infection, structural changes occur in the mammary gland 43. In particular, the presence of S. aureus in the mammary gland was shown to cause the size of the lumen to decrease, while the size of the stromal area increases 44. Furthermore, infected epithelial cells display less secretory activity compared to uninfected epithelial cells, while damage to the epithelial cells and areas of necrosis are caused by pathogen activity. Immune cells, such as neutrophils, macrophages and lymphocytes are also recruited to the infected epithelial lining in the alveolar lumen. Recently it was shown that S. aureus can induce autophagy in macrophages, while it may disrupt the autophagy pathway at a later stage. This will lead to an increased number of autophagosomes and eventually an increased survival of S. aureus in the respective macrophages 45. Autophagy is a mechanism, which is usually employed by cells to remove damaged or non-functional organelles and large proteins. However, this process can also facilitate the elimination of intracellular bacteria 46. Two recent studies have shown that S. aureus is capable of inducing and taking advantage of autophagy to survive within epithelial cells. Thus, it was shown that, whereas the formation of autophagosomes was increased, the fusion with lysosomes was interrupted 47,48. In both studies, it was observed that increased autophagy led to increased numbers of intracellular S. aureus cells, while decreased autophagy led to reduced numbers of intracellular S. aureus.
Once the presence of S. aureus is detected, cells in the mammary gland will change gene expression to produce molecules of the innate immune system 33. In a study performed with four tissues from different parts of the mammary gland of lactating cows, it was shown that the expression of Toll-like receptors 1-10 and NOD-like receptors 1-2, together with pro-inflammatory cytokines 6, IL-17A, IL-8 and the anti-inflammatory cytokine IL-10 was induced upon infection with S. aureus 33. Besides this, the expression of genes for acute phase proteins (APPs) and antimicrobial peptides (AMPs) was observed. The produced AMPs exert anti-microbial effects, while APPs are mainly involved in the regulation of early protective responses 49. The Toll-like receptors and NOD-like receptors
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are pattern recognition receptors (PRRs), which recognize invading pathogens50. The activation of PRRs usually leads to activation of the innate immune system, which includes the expression of APPs and AMPs. Most of the produced cytokines in the mammary tissue were pro-inflammatory, which means that they stimulated the inflammatory process. Nonetheless, the anti-inflammatory cytokine IL-10 was also produced, possibly to limit tissue damage in the gland, or due to an activity of S. aureus to avoid an active immune response 33.
8 Fig ure 1 . D ia gr am o f th e u dd er , s ho w in g s ch em ati ca lly h ow S . a ureu s c an c au se m as titis . S. a ur eus e nte rs the ud de r thro ug h the te at. It su bse que ntly rea ch es the e pit he lia l c ells prese nt in the a lv eo lus , w he re it w ill st art to d eve lo p a b io film . Le ukocy te s, su ch as ne utro phils and m acro pha ge s m igr ate into the a lv eo la r lu m en and sta rt to p ha go cyto se S. a ur eus . Som e S . aur eus c ells are ca pa ble o f su rviv ing ins id e ne ut ro phils. Ep ithe lia l ce lls w ill sta rt to p ro duce a ntim icro bia l a ge nts to co m ba t the p atho ge n. S. a ur eus is ho w eve r ca pa ble o f e nte rin g th e e pit he lia l ce lls. Som e e pit he lia l ce lls m ay be d am ag ed e ithe r b y the inf ecti on of S. a ur eus a nd /or the pro te ase s p ro duce d b y the ne utro phils, allowing t he b acte riu m to ca us e d ee pe r s ea te d inf ecti ons . (C rea te d w ith Bio R end er.co m )
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Virulence factors of S. aureus
S. aureus expresses several proteins that target different parts of the immune system to disable or counteract it, allowing the bacterium to thrive and survive in a generally hostile host environment 51,52. Such staphylococcal immune evasion proteins have been characterized in multiple studies 53,54, including studies on S. aureus strains of bovine origin (Wolf et al., 2011; Rocha et al., 2019). There are two particularly well studied S. aureus strains that are associated with bovine mastitis, namely S. aureus N305 and RF-122, and both are considered representative for most bovine mastitis cases worldwide 58. Several previous studies have tried to link particular virulence factors of S. aureus to the course of infection 54,59. For example, in a study performed in Sweden, it was shown that the genes for certain virulence factors, such as enterotoxins, hemolysins, leukocidins D and LukM/LukF-P83, clumping factors A and B, fibrinogen-binding protein and fibronectin-binding protein A, were more frequently found in S. aureus isolated from milk of cows with acute clinical mastitis 54. Enterotoxins are proteins which are secreted by S. aureus and that can act as superantigens on host immune cells 60. On the other hand, hemolysins are secretory molecules that are responsible for lysing red blood cells of the host 61. Leukocidins, such as leukocidin D and LukM/LukF-p83, are toxins that are secreted by S. aureus to induce pore formation in the membrane of the host leukocytes and kill them 62. The leukocidins LukM/LukF are exclusively detected in strains isolated from ruminants with mastitis 63. The virulence factors, clumping factors A and B, fibrinogen-binding protein and fibronectin-binding protein A are surface molecules that promote the adhesion of bacteria to the surface of host cells 54. Another study tried to link the occurrence of subclinical mastitis to a combination of S. aureus genes for virulence factors. Here, the spa and sej genes, which encode staphylococcal protein A and an enterotoxin respectively, were reported as potential risk factors for development of subclinical mastitis 59. Protein A is a surface protein of S. aureus, which abates the effects of host antibodies by binding to the fragment crystallizable (Fc) domain of immunoglobulins 64. However, despite the efforts of these studies, most virulence factors of S. aureus can currently not be linked to a specific type of infection.
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Interestingly, the loss of a single factor, the sigma factor SigB, was shown to have major consequences for the pathogenicity of S. aureus in relation to mastitis65. Here it should be noted that SigB regulates the expression of many genes in S. aureus and altered SigB-mediated gene expression can have a massive effect on S. aureus’ behavior 66–68. In particular, a gene called rsbU, which is part of the sigB locus, was observed to be mutated during a case of chronic subclinical mastitis. Within three months, the mutated S. aureus strain completely replaced the original S. aureus strain. This new strain showed different characteristics, such as increased proteolytic activity and biofilm formation, but was less virulent compared to the original strain. The new phenotypic traits were thought to be caused by a loss of SigB activity. This relates to the fact that RsbU, which in this case was mutated, has an important role in SigB regulation 69. In many conditions, SigB forms a complex with the anti-sigma factor RsbW, while the anti-anti sigma factor RsbV is inactivated by RsbW. This keeps SigB in an inactive state. However, under certain conditions, RsbV is activated by RsbU and, in turn, RsbV binds RsbW, leading to the release of SigB. The released SigB will then bind to RNA polymerase, resulting in the transcription of SigB-dependent genes. Thus, it seems that the inactive state of SigB due to mutation of rsbU resulted in a changed expression of many genes, which gave rise to an altered behavior that may unlock new nutritional resources and promote immune evasion by the respective S. aureus cells 65. The enterotoxin C (Sec) is the toxin most frequently produced by S. aureus strains isolated from bovine mastitis 60. After injection of the enterotoxin in mouse mammary glands, the injected tissue suffered from a severe damage and inflammation with infiltration by inflammatory cells. On top of that, the level of the IL-1β and IL-6 were significantly higher in the challenged tissue compared to the controls 60.
Another virulence factor often detected in S. aureus from bovine origin is the protease aureolysin. This is a secreted metalloprotease of S. aureus, which contributes to the immune evasion of S. aureus by cleaving the complement protein C3 in a different part than is normally done by the C3 convertases, which cleave the C3 protein into the C3a and C3b fragments, an essential step in the complement cascade 70. In this way, the bacteria can prevent their opsonization and posterior phagocytosis 57,71.
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The surface-associated serine-aspartate repeat protein E (SdrE) is a proteinthat specifically binds the complement control protein factor H, which inhibits the alternative pathway in the complement cascade 72. SdrE was found to be produced by many clinical bovine S. aureus isolates, including the prototype strains N305 and RF-122 57.
Other important virulence factors of S. aureus are the surface adhesins. These proteins are essential in the interaction of S. aureus with the host cells 73. Besides the already mention adhesins ClfA, ClfB and SdrE, also the iron-regulated surface determinants (IsdA, B, C and H) have been identified in strains of bovine origin 53,58. IsdA has been described to bind to fibronectin, fibrinogen, transferrin, hemoglobin, hemin and fetuin 74. IsdB, binds to hemoglobin and hemin, while IsdC is known to bind to hemin, and IsdH to haptoglobulin and the haptoglobulin–hemoglobin complex 73. Consistent with their role in iron acquisition, the proteins IsdA, IsdB and IsdH have been described to be more expressed in iron restricted environments 73,75.
The extracellular complement-binding protein (Ecb) and extracellular fibrinogen-binding protein (Efb) are homologous proteins 76. They block C5 convertases, which cleave the complement protein C5 into the C5a and C5b fragments. Subsequently, C5b will bind to the bacterial cell surface, serving as a platform for the so-called membrane attack complex 77. Blocking the C5 convertases results in inhibition of the complement cascade 71,76. Another role of Efb is to form a shield of host proteins around the bacterium, helping S. aureus to escape from phagocytosis 78. Also the Ecb and Efb proteins have been identified in strain RF-122 76 and multiple other bovine S. aureus isolates 57.
The staphylococcal superantigen-like (SSL) proteins are structurally similar to superantigens (SAgs), but serve a different function. SSL proteins generally bind to specific immune proteins, like IgGs and complement proteins 79. SSL1 from bovine S. aureus isolates was shown to inhibit the immune response by cleaving cytokines, like IFN-γ, IL-8 and IL-17A 79. Furthermore, the SSL6 blocks CD47, a protein present on erythrocytes, which protects erythrocytes against phagocytosis after inappropriate complement activation 80. SSL7 binds both IgA and the complement protein C5. This inhibits the complement cascade and the formation of a membrane attack complex. These proteins were also identified
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in some strains from bovine origin 56. Another SSL present in some bovine strains is SSL11, which acts directly on neutrophils. It induces strong cell adhesion, which makes neutrophils stick stronger to blood vessels, making it harder to migrate to a site of infection 81.
S. aureus antigens tested as vaccine targets
To protect cows from S. aureus-mediated mastitis, several studies have been performed in an attempt to create a vaccine against the mastitis-causing staphylococci. These potential vaccines are based on whole killed or attenuated bacteria, also referred to as bacterins, or on specific molecules that are produced by S. aureus 82,83. In this section an overview of S. aureus proteins is presented that have previously been tested as antigens for vaccination. In a study performed by Nelson et al., dairy cattle was vaccinated twice with a fusion protein of the fibronectin-binding protein A denoted zz-FnBPA, once during the non-lactation period and once more during lactation. After challenging the cows with intramammary S. aureus infusions, no difference was observed in the somatic cell counts (SCC) between the vaccinated and non-vaccinated cows. However, when the fusion protein zz-FnBPA was conjugated with immunostimulating complexes, higher antibody titers were measured and no cases of mastitis were detected after challenging the vaccinated cows with S. aureus, which was not the case in the non-vaccinated cows. Besides that, a lower count of SCC was observed in the vaccinated cows versus the non-vaccinated ones. On day 17 post-challenge, the counts of milk SCC were similar for the two groups, showing that no longer-term protection had been achieved in the vaccinated cows 84.
In a study performed in 2009, the antigenicity of a set of recombinant proteins from the iron-acquisition system (IsdH, SirA, FhuD2, SrtB, SstD, FeoB and IsdB) was measured in rabbits and cattle. The selection of these proteins was based on a previous study where the gene expression of S. aureus grown in tissue cages on mice was compared with gene expression of S. aureus grown in an iron-restricted medium 85. After injection of the recombinant proteins, all proteins showed antigenicity in the selected animal models. However, IsdH was the most promising candidate, showing a strong and long-lasting immune response in cattle with immunoglobulin G2 (IgG2) production 75.
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A well-studied staphylococcal virulence factor related to mastitis is theafore-mentioned leukocidin LukMF’50. A study with the intention to use this protein for diagnostics, showed that specific antibodies against LukM can provide protection against mastitis. Two variants of antibodies against the C-terminus of LukM were produced, and both were able to deactivate LukM. The C-terminus of LukM entails the amino acids that are needed to connect to the cell membrane of immune cells. Consequently, the antibody-mediated deactivation of LukM prevented LukMF’ from binding to neutrophils and from killing the targeted neutrophils through pore formation 86.
Another virulence factor used to create a potential vaccine against mastitis was SEC 82,87. Two studies tested the protective effect of two different mutated SEC molecules against experimental bovine mastitis caused by S. aureus. The first study showed that immunization with a mutant SEC protein led to higher levels of antibodies against SEC in blood samples, resulting in lowered SCCs compared to non-vaccinated cows. Milk samples from immunized cows contained no detectable S. aureus, while S. aureus was detected in 75% of the samples from non-vaccinated cows 87. In 2010, the effect of a different SEC mutant protein, which was linked to the glutathione S-transferase (GST) was tested 82. Immunization with this mutant GST-SEC also led to lower SCCs in milk samples compared to non-vaccinated cows. On top of this, the level of SEC-specific antibodies in blood and milk samples increased as a consequence of the immunization 82. Therefore, the results of these studies suggested that immunization with mutant SEC proteins elicits protective immune responses against S. aureus-induced mastitis.
In a recent study, young heifers naive for S. aureus, were vaccinated with two S. aureus immune evasion proteins, namely Efb and LukM. After three subcutaneous immunizations an increment in antibody levels was observed against the tested proteins in serum, colostrum, and milk compared to animals naturally exposed to S. aureus. In addition, significantly higher levels of the interleukin IL17 were identified in the serum of the vaccinated cows 88.
Lastly, Merril et al. used a set of S. aureus surface proteins (SASP) and Staphylococcus chromogenes surface proteins (SCSP) to vaccinate a group of Holstein dairy cows. The cows were vaccinated thrice, at 28 and 14 days before drying off, and at dry off. Interestingly, after challenging all cows with S. aureus
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culture suspensions, only one out of six cows vaccinated with the SASP was protected against staphylococcal mastitis. On the other hand, the group of cows vaccinated with SCSP developed neither subclinical nor clinical mastitis 89. The result of this study suggests that the employment of SCPS, which results in a cross-protection against S. aureus mastitis could represent an effective vaccination approach to control staphylococcal mastitis in bovines.
Scope of the thesis
This PhD thesis describes the impact and consequences of mastitis for cows and the dairy industry, and it addresses possible avenues towards the development of anti-staphylococcal vaccines. Special attention has been attributed to the analysis of S. aureus isolates from Mexican cows with mastitis because, at the start of this PhD research project, very little was known about their diversity in terms of lineages and the virulence factors they produce. A brief description of the types and etiological agents of mastitis in dairy cows is presented in the introductory Chapter 1. In addition, this chapter addresses the immunology of the mammary gland and the pathology of mastitis due to infection with S. aureus. The chapter also presents an overview of the various known and studied virulence factors that S. aureus employs to cause infection in the udder. Chapter 1 concludes with an overview of the proteins that have been tested as antigens for vaccination in bovine mastitis models. A general overview of the studies presented in the different thesis chapters, and the respective approaches is presented in Figure 2.
Chapter 2 of this thesis describes the typing and antibiotic susceptibility testing of S. aureus isolates causing bovine mastitis in Mexico. In total, 33 S aureus isolates were collected from milk samples. To assess the relatedness of these isolates, multiple-locus variable number tandem repeat fingerprinting (MLVF) was used, showing that they belong to four different MLVF clusters. In addition, spa-typing was performed to complement the MLVF analysis, showing the presence of four different spa-types (t224, t3196, t416 and t114). Variations in the respective masses of Protein A, which is encoded by the spa gene, were confirmed by Western blotting analysis. Also, the absence of a PCR fragment for the sdrE gene suggested by MLVF was confirmed by Western blotting. Antibiotic sensitivity tests showed that 32 out of the 33 isolates were resistant
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against benzylpenicillin, only 2 strains were resistant against chloramphenicol,and one strain was susceptible for all the tested antibiotics. Many of the S. aureus isolates carried a unique plasmid of 1502 bp designated as pSAM1. Altogether, the results presented in this chapter suggest that, at the time of sampling, only a few lineages of S. aureus were responsible for causing mastitis in Mexican cows of the Comarca Lagunera, and that the majority of the isolates were resistant against benzylpenicillin. The latter finding is consistent with the fact that this antibiotic is frequently used to treat mastitis.
Chapter 3 of this thesis presents a surfacome analysis of six S. aureus isolates from mastitic milk. As outlined in Chapter 1, surface proteins are essential virulence factors that mediate the interactions between the pathogen and the cells and tissues of the host. Cell surface-associated proteins are therefore considered as important potential targets for vaccines. In silico prediction of exported proteins in combination with the actual identification of the cell surface proteins were employed to analyze the surfacome of the investigated strains. For the extraction of the cell surface proteins, S aureus cells were grown in whey permeate in order to mimic the environment inside the udder. Subsequently, the cells were treated either with immobilized trypsin to shave proteins from the surface of the S. aureus cells, or with potassium thiocyanide (KSCN) to extract non-covalently cell wall-bound proteins. A total number of 258 proteins was identified, with an overlap of 28.3% for the two methods of extraction. Interestingly, each investigated isolate displayed a characteristic set of surface proteins. Nevertheless, the results of this study highlight several cell surface-exposed proteins of S. aureus as potential vaccine targets for the prevention of mastitis in cows.
A group of cell wall-bound S. aureus proteins that could represent relevant vaccine targets are peptidoglycan hydrolases. Therefore, the studies described in Chapter 4 were aimed at assessing the immunogenicity of the non-covalently cell wall-associated peptidoglycan hydrolases Sle1, Aly and LytM of S. aureus. In particular, this involved a comparative analysis of the binding of human and murine IgGs to the purified full-size Sle1, Aly and LytM proteins, or to different domains of which these enzymes are composed. To this end, the proteins and their domains were expressed in Lactococcus lactis and subsequently purified. The quality of the purified proteins was assessed by measuring their biological activity. Immunization of mice showed that the purified full-size proteins elicited
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specific IgGs, but these IgGs did not protect the mice from death by S. aureus bacteremia. To obtain a better understanding of the diversity in target epitopes of Sle1, Aly and LytM, we compared the binding of serum IgGs from healthy human volunteers, highly S. aureus-colonized patients with the genetic blistering disease epidermolysis bullosa, or the immunized mice to the purified full-size proteins and their different domains. The results show that the most abundant serum IgGs target the cell wall-binding domain of Sle1, and the catalytic domains of Aly and LytM. However, as shown by the murine infection model, these particular IgGs were not protective against S. aureus bacteremia. In contrast, less abundant IgGs against the catalytic domain of Sle1 and the N-terminal domains of Aly and LytM were almost exclusively detected in humans, where they may contribute to protection against staphylococcal infections. Together, these observations focus attention on the use of particular protein domains for vaccination rather than the respective full-size proteins. This could help to direct potentially protective immune responses towards the most promising epitopes within staphylococcal antigens, and lead to effective anti-S. aureus vaccines.
Chapter 5 of this thesis summarizes the main findings and conclusions of the presented studies. In addition, the results are discussed and perspectives for future research are outlined.
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Figure 2. Schematic representation of the experimental pipeline described in this thesis to identify staphylococcal cell wall antigen domains for potential use as vaccine targets. Identification and typing of S. aureus isolates from milk samples obtained from cows suffering from clinical mastitis was performed by MLVF and spa-typing. The subsequent identification of surface-exposed or cell wall-located proteins (i.e. the surfacome) from pathogenic S. aureus isolates was achieved by proteomics, using cell surface shaving and cell wall protein extraction. 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, which were subsequently challenged with S. aureus in a bacteremia model. Finally, several antigen domains were used for the mapping of epitopes that are recognized by the IgGs from immunized mice and cows suffering from mastitis.
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References
1. Contreras, G. A. & Rodríguez, J. M. Mastitis: Comparative Etiology and Epidemiology. J. Mammary Gland Biol. Neoplasia 16, 339–356 (2011). 2. Hand, K. J., Godkin, A. & Kelton, D. F. Milk production and somatic cell
counts: A cow-level analysis. J. Dairy Sci. 93, 1358–1362 (2012).
3. Raboisson, D. et al. The Use of Meta-Analysis for the Measurement of Animal Disease Burden: Losses Due to Clinical Mastitis as an Example. Front. Vet. Sci. 7, (2020).
4. van Soest, F. J. S., Santman-Berends, I. M. G. A., Lam, T. J. G. M. & Hogeveen, H. Failure and preventive costs of mastitis on Dutch dairy farms. J. Dairy Sci. 99, 8365–8374 (2016).
5. Olde Riekerink, R. G. M., Barkema, H. W., Kelton, D. F. & Scholl, D. T. Incidence Rate of Clinical Mastitis on Canadian Dairy Farms. J. Dairy Sci. 91, 1366–1377 (2008).
6. Sutra, L. & Poutrel, B. Virulence factors involved in the pathogenesis of bovine intramammary infections due to Staphylococcus aureus. J. Med. Microbiol. 40, 79–89 (1994).
7. Weese, J. S. & van Duijkeren, E. Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in veterinary medicine. Veterinary Microbiology 140, 418–429 (2010).
8. Sibbald, M. J. J. B. et al. Mapping the Pathways to StaphylococcalPathogenesis by Comparative Secretomics. Microbiol. Mol. Biol. Rev. 70, 755–788 (2006).
9. Ziebandt, A.-K. et al. Proteomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene regulation. Proteomics 10, 1634–1644 (2010).
10. Sharma, A. K. et al. Bacterial Virulence Factors: Secreted for Survival. Indian J. Microbiol. 57, 1–10 (2017).
11. Dinges, M. M., Orwin, P. M. & Schlievert, P. M. Exotoxins of Staphylococcus aureus. Clin. Microbiol. Rev. 13, 16–34 (2000).
12. Schlievert, P. M., Strandberg, K. L., Lin, Y.-C., Peterson, M. L. & Leung, D. Y. M. Secreted virulence factor comparison between methicillin-resistant and methicillin-sensitive Staphylococcus aureus, and its relevance to atopic dermatitis. J. Allergy Clin. Immunol. 125, 39–49 (2010).
13. van Trijp, M. J. C. A. et al. Genotypes, superantigen gene profiles, and presence of exfoliative toxin genes in clinical methicillin-susceptible Staphylococcus aureus isolates. Diagn. Microbiol. Infect. Dis. 66, 222–224 (2010).
19
1
14. Palma Medina, L. M. et al. Metabolic Cross-talk Between Human BronchialEpithelial Cells and Internalized Staphylococcus aureus as a Driver for Infection. Mol. Cell. Proteomics 18, 892–908 (2019).
15. Mekonnen, S. A. et al. Metabolic niche adaptation of community- and hospital-associated methicillin-resistant Staphylococcus aureus. J. Proteomics 193, 154–161 (2019).
16. Rainard, P. et al. Knowledge gaps and research priorities in Staphylococcus aureus mastitis control. Transbound. Emerg. Dis. 65, 149–165 (2018). 17. Sordillo, L. M. & Streicher, K. L. Mammary gland immunity and mastitis
susceptibility. J. Mammary Gland Biol. Neoplasia 7, 135–146 (2002). 18. Capuco, A. V. et al. Increased Susceptibility to lntramammary Infection
Following Removal of Teat Canal Keratin. J. Dairy Sci. 75, 2126–2130 (1992).
19. Paape, M. J., Shafer-Weaver, K., Capuco, A. V., Van Oostveldt, K. & Burvenich, C. Immune Surveillance of Mammary Tissue by Phagocytic Cells. in Biology of the Mammary Gland. Advances in Experimental Medicine and Biology (eds. Mol, J. A. & Clegg, R. A.) 259–277 (Springer, Boston, MA, 2002).
20. Mehrzad, J., Paape, M. & Burvenich, C. Role of neutrophils in protection of udder from infection in high yielding dairy cows. Iran. J. Vet. Res. 11, 102– 118 (2010).
21. Persson, K., Larsson, I. & Hallén Sandgren, C. Effects of certain inflammatory mediators on bovine neutrophil migration in vivo and in vitro. Vet. Immunol. Immunopathol. 37, 99–112 (1993).
22. Fitzpatrick, J. L., Cripps, P. J., Hill, A. W., Bland, P. W. & Stokes, C. R. MHC class II expression in the bovine mammary gland. Vet. Immunol. Immunopathol. 32, 13–23 (1992).
23. Park, Y. H., Fox, L. K., Hamilton, M. J. & Davis, W. C. Suppression of proliferative response of BoCD4+ T lymphocytes by activated BoCD8+ T lymphocytes in the mammary gland of cows with Staphylococcus aureus mastitis. Vet. Immunol. Immunopathol. 36, 137–151 (1993).
24. Ezzat Alnakip, M. et al. The Immunology of Mammary Gland of Dairy Ruminants between Healthy and Inflammatory Conditions. J. Vet. Med. 2014, 1–31 (2014).
25. Kitano, N., Isobe, N., Noda, J. & Takahashi, T. Concentration patterns of antibacterial factors and immunoglobulin A antibody in foremilk fractions of healthy cows. Anim. Sci. J. 91, (2020).
26. Huang, Y. Q., Morimoto, K., Hosoda, K., Yoshimura, Y. & Isobe, N. Differential immunolocalization between lingual antimicrobial peptide and
20
lactoferrin in mammary gland of dairy cows. Vet. Immunol. Immunopathol. 145, 499–504 (2012).
27. Rainard, P. The complement in milk and defense of the bovine mammary gland against infections. Vet. Res. 34, 647–670 (2003).
28. Griesbeck-Zilch, B., Meyer, H. H. D., Kühn, C., Schwerin, M. & Wellnitz, O. Staphylococcus aureus and Escherichia coli Cause Deviating Expression Profiles of Cytokines and Lactoferrin Messenger Ribonucleic Acid in Mammary Epithelial Cells. J. Dairy Sci. 91, 2215–2224 (2008).
29. Barrio, M. B., Rainard, P. & Poutrel, B. Milk complement and the opsonophagocytosis and killing of Staphylococcus aureus mastitis isolates by bovine neutrophils. Microb. Pathog. 34, 1–9 (2003).
30. Frank, M. M. & Fries, L. F. The role of complement in inflammation and phagocytosis. Immunol. Today 12, 322–326 (1991).
31. Carroll, M. C. Complement and humoral immunity. Vaccine 26, I28–I33 (2008).
32. Lamkanfi, M. & Dixit, V. M. Inflammasomes and Their Roles in Health and Disease. Annu. Rev. Cell Dev. Biol. 28, 137–161 (2012).
33. Whelehan, C. J., Meade, K. G., Eckersall, P. D., Young, F. J. & O’Farrelly, C. Experimental Staphylococcus aureus infection of the mammary gland induces region-specific changes in innate immune gene expression. Vet. Immunol. Immunopathol. 140, 181–189 (2011).
34. Oliveira, M. et al. Time course of biofilm formation by Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates. Vet. Microbiol. 124, 187– 191 (2007).
35. Zhang, L. et al. Virulence gene profiles: alpha-hemolysin and clonal diversity in Staphylococcus aureus isolates from bovine clinical mastitis in China. BMC Vet. Res. 14, 63 (2018).
36. Melchior, M. B., Vaarkamp, H. & Fink-Gremmels, J. Biofilms: A role in recurrent mastitis infections? Vet. J. 171, 398–407 (2006).
37. Peterson, B. W. et al. Viscoelasticity of biofilms and their recalcitrance to mechanical and chemical challenges. FEMS Microbiol. Rev. 39, 234–245 (2015).
38. Gotz, F. Staphylococcus and biofilms. Mol. Microbiol. 43, 1367–1378 (2002).
39. Melchior, M. B., Fink-Gremmels, J. & Gaastra, W. Comparative Assessment of the Antimicrobial Susceptibility of Staphylococcus aureus Isolates from Bovine Mastitis in Biofilm Versus Planktonic Culture. J. Vet. Med. Ser. B 53, 326–332 (2006).
21
1
40. Palma Medina, L. M. et al. Interaction of Staphylococcus aureus and HostCells upon Infection of Bronchial Epithelium during Different Stages of Regeneration. ACS Infect. Dis. 6, 2279–2290 (2020).
41. Mekonnen, S. A. et al. Signatures of cytoplasmic proteins in the exoproteome distinguish community- and hospital-associated methicillin-resistant Staphylococcus aureus USA300 lineages. Virulence 8, 891–907 (2017). 42. Zhang, M. et al. TGF-β1 promoted the infection of bovine mammary epithelial
cells by Staphylococcus aureus through increasing expression of cells’ fibronectin and integrin β1. Vet. Microbiol. 237, 108420 (2019).
43. Bianchi, R. M. et al. Pathological and microbiological characterization of mastitis in dairy cows. Trop. Anim. Health Prod. 51, 2057–2066 (2019). 44. Sordillo, L. M., Nickerson, S. C. & Akers, R. M. Pathology of Staphylococcus
aureus during Lactogenesis: Relationships with Bovine Mammary Structure and Function. J. Dairy Sci. 72, 228–240 (1989).
45. Cai, J. et al. Staphylococcus aureus facilitates its survival in bovine macrophages by blocking autophagic flux. J. Cell. Mol. Med. 24, 3460–3468 (2020).
46. Rich, K. A., Burkett, C. & Webster, P. Cytoplasmic bacteria can be targets for autophagy. Cell. Microbiol. 5, 455–468 (2003).
47. Wang, H. et al. Staphylococcus aureus induces autophagy in bovine mammary epithelial cells and the formation of autophagosomes facilitates intracellular replication of S. aureus. J. Dairy Sci. 102, 8264–8272 (2019). 48. Geng, N. et al. Autophagy of bovine mammary epithelial cell induced by
intracellular Staphylococcus aureus. J. Microbiol. 58, 320–329 (2020). 49. Rainard, P. & Riollet, C. Innate immunity of the bovine mammary gland. Vet.
Res. 37, 369–400 (2006).
50. Fukata, M., Vamadevan, A. S. & Abreu, M. T. Toll-like receptors (TLRs) and Nod-like receptors (NLRs) in inflammatory disorders. Semin. Immunol. 21, 242–253 (2009).
51. Fedtke, I., Götz, F. & Peschel, A. Bacterial evasion of innate host defenses – the Staphylococcus aureus lesson. Int. J. Med. Microbiol. 294, 189–194 (2004).
52. Buchan, K. D., Foster, S. J. & Renshaw, S. A. Staphylococcus aureus: setting its sights on the human innate immune system. Microbiology 165, 367–385 (2019).
53. Zecconi, A. & Scali, F. Staphylococcus aureus virulence factors in evasion from innate immune defenses in human and animal diseases. Immunol. Lett. 150, 12–22 (2013).
22
54. Artursson, K., Söderlund, R., Liu, L., Monecke, S. & Schelin, J. Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics. Vet. Microbiol. 193, 156–161 (2016).
55. McCarthy, A. J. & Lindsay, J. A. Genetic variation in Staphylococcus aureus surface and immune evasion genes is lineage associated: Implications for vaccine design and host-pathogen interactions. BMC Microbiol. 10, 173 (2010).
56. Wolf, C. et al. Genomic and proteomic characterization of Staphylococcus aureus mastitis isolates of bovine origin. Proteomics 11, 2491–2502 (2011). 57. Rocha, L. S. et al. Comparative genomics of Staphylococcus aureus associated with subclinical and clinical bovine mastitis. PLoS One 14, e0220804 (2019).
58. Peton, V. et al. Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines. Vet. Res. 45, 106 (2014).
59. Zecconi, A., Cesaris, L., Liandris, E., Daprà, V. & Piccinini, R. Role of several Staphylococcus aureus virulence factors on the inflammatory response in bovine mammary gland. Microb. Pathog. 40, 177–183 (2006).
60. Fang, R. et al. Staphylococcal Enterotoxin C Is an Important Virulence Factor for Mastitis. Toxins (Basel). 11, 141 (2019).
61. P. Skaar, E. & Schneewind, O. Iron-regulated surface determinants (Isd) of Staphylococcus aureus: stealing iron from heme. Microbes Infect. 6, 390– 397 (2004).
62. Vrieling, M. et al. Bovine Staphylococcus aureus Secretes the Leukocidin LukMF′ To Kill Migrating Neutrophils through CCR1. MBio 6, (2015). 63. Barrio, M. B., Rainard, P. & Prévost, G. LukM/LukF’-PV is the most active
Staphylococcus aureus leukotoxin on bovine neutrophils. Microbes Infect. 8, 2068–2074 (2006).
64. Falugi, F., Kim, H. K., Missiakas, D. M. & Schneewind, O. Role of Protein A in the Evasion of Host Adaptive Immune Responses by Staphylococcus aureus. MBio 4, (2013).
65. Marbach, H. et al. Within-host evolution of bovine Staphylococcus aureus selects for a SigB-deficient pathotype characterized by reduced virulence but enhanced proteolytic activity and biofilm formation. Sci. Rep. 9, 13479 (2019).
66. Mäder, U. et al. Staphylococcus aureus Transcriptome Architecture: From Laboratory to Infection-Mimicking Conditions. PLOS Genet. 12, e1005962 (2016).
23
1
aureus Adaptation during Chronic Infections. PLOS Pathog. 11, e1004870(2015).
68. Pförtner, H. et al. Activation of the alternative sigma factor SigB of Staphylococcus aureus following internalization by epithelial cells – An in vivo proteomics perspective. Int. J. Med. Microbiol. 304, 177–187 (2014). 69. Hecker, M., Pané-Farré, J. & Uwe, V. SigB-Dependent General Stress
Response in Bacillus subtilis and Related Gram-Positive Bacteria. Annu. Rev. Microbiol. 61, 215–236 (2007).
70. Rooijakkers, S. H. M. & van Strijp, J. A. G. Bacterial complement evasion. Mol. Immunol. 44, 23–32 (2007).
71. de Jong, N. W. M., van Kessel, K. P. M. & van Strijp, J. A. G. Immune Evasion by Staphylococcus aureus. Microbiol. Spectr. 7, 1–27 (2019).
72. Sharp, J. A. et al. Staphylococcus aureus Surface Protein SdrE Binds Complement Regulator Factor H as an Immune Evasion Tactic. PLoS One 7, e38407 (2012).
73. Clarke, S. R. & Foster, S. J. Surface Adhesins of Staphylococcus aureus. in Advances in Microbial Physiology 51, 187–224 (2006).
74. Speziale, P. et al. Structural and functional role of Staphylococcus aureus surface components recognizing adhesive matrix molecules of the host. Future Microbiol. 4, 1337–1352 (2009).
75. Ster, C. et al. Evaluation of some Staphylococcus aureus iron-regulated proteins as vaccine targets. Vet. Immunol. Immunopathol. 136, 311–318 (2010).
76. Jongerius, I. et al. Staphylococcal complement evasion by various convertase-blocking molecules. J. Exp. Med. 204, 2461–2471 (2007). 77. Complement. in Immunology for Pharmacy (ed. Flaherty, D. K. B. T.-I. for P.)
87–96 (Elsevier, 2012).
78. Ko, Y.-P. et al. Phagocytosis Escape by a Staphylococcus aureus Protein That Connects Complement and Coagulation Proteins at the Bacterial Surface. PLoS Pathog. 9, e1003816 (2013).
79. Tang, A. et al. Staphylococcus aureus Superantigen-Like Protein SSL1: A Toxic Protease. Pathogens 8, 2 (2019).
80. Fevre, C. et al. Staphylococcus aureus proteins SSL6 and SElX interact with neutrophil receptors as identified using secretome phage display. Cell. Microbiol. 16, 1646–1665 (2014).
81. Chen, C., Yang, C. & Barbieri, J. T. Staphylococcal Superantigen-like protein 11 mediates neutrophil adhesion and motility arrest, a unique bacterial toxin action. Sci. Rep. 9, 4211 (2019).
24
82. Cui, J.-C. et al. Protective effect of glutathione S-transferase-fused mutant staphylococcal enterotoxin C against Staphylococcus aureus-induced bovine mastitis. Vet. Immunol. Immunopathol. 135, 64–70 (2010).
83. Côté-Gravel, J., Brouillette, E. & Malouin, F. Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses against Staphylococcus aureus. PLoS One 14, e0227109 (2019). 84. Nelson, L. et al. Adhesins in staphylococcal mastitis as vaccine components.
Flem. Vet. J. 62, 111–125 (1990).
85. Allard, M. et al. Transcriptional modulation of some Staphylococcus aureus iron-regulated genes during growth in vitro and in a tissue cage model in vivo. Microbes Infect. 8, 1679–1690 (2006).
86. Padmaja, R. J. & Halami, P. M. Taming C-terminal peptides of Staphylococcus aureus leukotoxin M for B-cell response: Implication in improved subclinical bovine mastitis diagnosis and protective efficacy in vitro. Toxicon 119, 99–105 (2016).
87. Chang, B. S. et al. Protective effects of recombinant staphylococcal enterotoxin type C mutant vaccine against experimental bovine infection by a strain of Staphylococcus aureus isolated from subclinical mastitis in dairy cattle. Vaccine 26, 2081–2091 (2008).
88. Benedictus, L. et al. Immunization of young heifers with staphylococcal immune evasion proteins before natural exposure to Staphylococcus aureus induces a humoral immune response in serum and milk. BMC Vet. Res. 15, 15 (2019).
89. Merrill, C. et al. Immunological responses and evaluation of the protection in dairy cows vaccinated with staphylococcal surface proteins. Vet. Immunol. Immunopathol. 214, 109890 (2019).
