Differential Response of Human Dendritic Cells upon Stimulation with Encapsulated or Non-Encapsulated Isogenic Strains of Porphyromonas gingivalis.

During periodontitis, the extracellular capsule of Porphyromonas gingivalis favors alveolar bone loss by inducing Th1 and Th17 patterns of lymphocyte response in the infected periodontium. Dendritic cells recognize bacterial antigens and present them to T lymphocytes, defining their activation and polarization. Thus, dendritic cells could be involved in the Th1 and Th17 response induced against the P. gingivalis capsule. Herein, monocyte-derived dendritic cells were obtained from healthy individuals and then stimulated with different encapsulated strains of P. gingivalis or two non-encapsulated isogenic mutants. Dendritic cell differentiation and maturation were analyzed by flow cytometry. The mRNA expression levels for distinct Th1-, Th17-, or T-regulatory-related cytokines and transcription factors, as well as TLR2 and TLR4, were assessed by qPCR. In addition, the production of IL-1ß, IL-6, IL-23, and TNF-α was analyzed by ELISA. The encapsulated strains and non-encapsulated mutants of P. gingivalis induced dendritic cell maturation to a similar extent; however, the pattern of dendritic cell response was different. In particular, the encapsulated strains of P. gingivalis induced higher expression of IRF4 and NOTCH2 and production of IL-1ß, IL-6, IL-23, and TNF-α compared with the non-encapsulated mutants, and thus, they showed an increased capacity to trigger Th1 and Th17-type responses in human dendritic cells.

Acapsular Staphylococcus aureus with a non-functional agr regains capsule expression after passage through the bloodstream in a bacteremia mouse model.

Selection pressures exerted on Staphylococcus aureus by host factors during infection may lead to the emergence of regulatory phenotypes better adapted to the infection site. Traits convenient for persistence may be fixed by mutation thus turning these mutants into microevolution endpoints. The feasibility that stable, non-encapsulated S. aureus mutants can regain expression of key virulence factors for survival in the bloodstream was investigated. S. aureus agr mutant HU-14 (IS256 insertion in agrC) from a patient with chronic osteomyelitis was passed through the bloodstream using a bacteriemia mouse model and derivative P3.1 was obtained. Although IS256 remained inserted in agrC, P3.1 regained production of capsular polysaccharide type 5 (CP5) and staphyloxanthin. Furthermore, P3.1 expressed higher levels of asp23/SigB when compared with parental strain HU-14. Strain P3.1 displayed decreased osteoclastogenesis capacity, thus indicating decreased adaptability to bone compared with strain HU-14 and exhibited a trend to be more virulent than parental strain HU-14. Strain P3.1 exhibited the loss of one IS256 copy, which was originally located in the HU-14 noncoding region between dnaG (DNA primase) and rpoD (sigA). This loss may be associated with the observed phenotype change but the mechanism remains unknown. In conclusion, S. aureus organisms that escape the infected bone may recover the expression of key virulence factors through a rapid microevolution pathway involving SigB regulation of key virulence factors.

Plasmid gene AZOBR_p60126 impacts biosynthesis of lipopolysaccharide II and swarming motility in Azospirillum brasilense Sp245.

The facultative plant endophyte Azospirillum brasilense Sp245 synthesizes two high-molecular-weight lipopolysaccharides, LPSI and LPSII, which comprise identical d-rhamnan O-polysaccharides and, presumably different core oligosaccharides. Previously, using random insertion mutagenesis, we constructed the LpsII- mutant KM139 of strain Sp245 that possessed an Omegon-Km insertion in plasmid AZOBR_p6. Here, we found that in KM139, Omegon-Km disrupted the coding sequence AZOBR_p60126 for a putative glycosyltransferase related to mannosyltransferases and rhamnosyltransferases. To verify its function, we cloned the AZOBR_p60126 gene of strain Sp245 in the expression vector plasmid pRK415 and transferred the construct pRK415-p60126 into KM139. In the complemented mutant KM139 (pRK415-p60126), the wild-type LPSI+ LPSII+ profile was recovered. We also compared the swimming and swarming motilities of strains Sp245, Sp245 (pRK415), KM139, KM139 (pRK415), and KM139 (pRK415-p60126). All these strains had the same flagellar-dependent swimming speeds, but on soft media, the LpsI+ LpsII- strains KM139 and KM139 (pRK415) swarmed significantly faster than the other LpsI+ LpsII+ strains. Such interstrain differences in swarming motility were more pronounced on 0.4% than on 0.5% soft agar plates. These data show that the AZOBR_p60126-encoded putative glycosyltransferase significantly affects the lipopolysaccharide profile and, as a consequence, the social motility of azospirilla.

The Interaction of Klebsiella pneumoniae With Lipid Rafts-Associated Cholesterol Increases Macrophage-Mediated Phagocytosis Due to Down Regulation of the Capsule Polysaccharide.

Klebsiella pneumoniae successfully colonizes host tissues by recognizing and interacting with cholesterol present on membrane-associated lipid rafts. In this study, we evaluated the role of cholesterol in the expression of capsule polysaccharide genes of K. pneumoniae and its implication in resistance to phagocytosis. Our data revealed that exogenous cholesterol added to K. pneumoniae increases macrophage-mediated phagocytosis. To explain this event, the expression of capsular galF, wzi, and manC genes was determined in the presence of cholesterol. Down-regulation of these capsular genes occurred leading to increased susceptibility to phagocytosis by macrophages. In contrast, depletion of cholesterol from macrophage membranes led to enhanced expression of galF, wzi, and manC genes and to capsule production resulting in resistance to macrophage-mediated phagocytosis. Cholesterol-mediated repression of capsular genes was dependent on the RcsA and H-NS global regulators. Finally, cholesterol also down-regulated the expression of genes responsible for LPS core oligosaccharides production and OMPs. Our results suggest that cholesterol plays an important role for the host by reducing the anti-phagocytic properties of the K. pneumoniae capsule facilitating bacterial engulfment by macrophages during the bacteria-eukaryotic cell interaction mediated by lipid rafts.

Phenotypic characterization of Cronobacter spp. strains isolated from foods and clinical specimens in Brazil.

Several Cronobacter species are opportunistic pathogens that cause infections in humans. This study evaluated the phenotypic characteristics of 57 Cronobacter strains (C. sakazakii n=41, C. malonaticus n=10, C. dublinensis n=4, and C. muytjensii n=2) isolated from food (n=54) and clinical specimens (n=3) in Brazil. These strains included sequence types (ST): ST395-ST398, ST402, ST413 and ST433-ST439, isolated from food samples, and three C. malonaticus clinical strains previous isolated from an outbreak which were ST394 (n=1) and ST440 (n=2). Strains were tested for capsule production, biofilm formation, protease activity, hemolytic activity, cell-cell aggregation, and desiccation resistance. Capsule formation was observed with all Cronobacter strains. Forty-four (77.2%) strains showed proteolytic activity on milk agar. All strains showed ß-hemolysis against erythrocytes from guinea pig, horse and rabbit. Using erythrocytes from sheep, the majority of strains (53/57; 92.9%) showed α-hemolysis and the remaining, ß-hemolysis. All Cronobacter strains produced weak biofilms in microtiters polystyrene plates, which were independent of temperature (4, 25 and 37°C) and/or growth conditions. In glass tubes, formation of either a moderate or strong biofilm was observed in 15/57 (26.3%), 19/57 (33.3%) and 27/57 (47.4%), at 4, 25 and 37°C, respectively. Desiccation treatment decreased Cronobacter viability by 1.55 to >3.87Log10CFU/mL. Cell-cell aggregation was observed in 17 (29.8%) strains. This study showed that the Cronobacter species evaluated showed differing phenotypes, independent of their origin (clinical or not) and ST. Further studies are necessary to elucidate the factors affecting phenotype expression. This may identify novel bacterial targets that could be useful in the development of strategies to control Cronobacter in food chain and to prevent cases of infections.

Lactic Acid Bacteria from Andean Grain Amaranth: A Source of Vitamins and Functional Value Enzymes.

Amaranth is a rediscovered pseudocereal with high nutritional properties. Lactic acid fermentation can increase the functional and nutritional value of foods. The aims of this study were to isolate and evaluate the functionality of lactic acid bacteria (LAB) from amaranth. LAB strains (n = 29) isolated from amaranth sourdough and grains included Lactobacillus (L.) plantarum (n = 8), L. rhamnosus (n = 6), Enterococcus (E.) mundtii (n = 4), E. hermanniensis (n = 3), E. durans (n = 1), Enterococcus sp. (n = 1), Leuconostoc (Lc.) mesenteroides (n = 3), and Lc. mesenteroides subsp. mesenteroides (n = 3). Only 21% of the strains showed the ability to synthesize capsular exopolysaccharides or display ropiness and only 8 strains showed amylolytic activity. L. plantarum CRL 2106 and E. durans CRL 2122 showed the highest phytase activity, which is of importance for mineral bioavailability. L. plantarum CRL 2106 and CRL 2107 and Lc. mesenteroides subsp. mesenteroides CRL 2131 synthesized the highest concentrations of B2 and B9 vitamin (140-250 ng/mL). This study demonstrates the potential of LAB to improve the nutritional and functional values of pseudocereal-derived foods.

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium.

Haemophilus influenzae type b (Hib), a Gram-negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl-ribitol-phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake-flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (∼500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α-ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508-1519, 2017.

Phosphorus-rich structures and capsular architecture in Cryptococcus neoformans.

AIM: In this study, we aimed to analyze the relationship of phosphorus-rich structures with surface architecture in Cryptococcus neoformans. METHODS: Phosphorus-rich structures in C. neoformans were analyzed by combining fluorescence microscopy, biochemical extraction, scanning electron microscopy, electron probe x-ray microanalysis and 3D reconstruction of high pressure frozen and freeze substituted cells by focused ion beam-scanning electron microscopy (FIB-SEM). RESULTS & CONCLUSION: Intracellular and surface phosphorus-enriched structures were identified. These molecules were required for capsule assembly, as demonstrated in experiments using polysaccharide incorporation by capsule-deficient cells and mutants with defects in polyphosphate synthesis. The demonstration of intracellular and cell wall-associated polyphosphates in C. neoformans may lead to future studies involving their participation in both physiologic and pathogenic events.

Pathogenic diversity amongst serotype C VGIII and VGIV Cryptococcus gattii isolates.

Cryptococcus gattii is one of the causative agents of human cryptococcosis. Highly virulent strains of serotype B C. gattii have been studied in detail, but little information is available on the pathogenic properties of serotype C isolates. In this study, we analyzed pathogenic determinants in three serotype C C. gattii isolates (106.97, ATCC 24066 and WM 779). Isolate ATCC 24066 (molecular type VGIII) differed from isolates WM 779 and 106.97 (both VGIV) in capsule dimensions, expression of CAP genes, chitooligomer distribution, and induction of host chitinase activity. Isolate WM 779 was more efficient than the others in producing pigments and all three isolates had distinct patterns of reactivity with antibodies to glucuronoxylomannan. This great phenotypic diversity reflected in differential pathogenicity. VGIV isolates WM 779 and 106.97 were similar in their ability to cause lethality and produced higher pulmonary fungal burden in a murine model of cryptococcosis, while isolate ATCC 24066 (VGIII) was unable to reach the brain and caused reduced lethality in intranasally infected mice. These results demonstrate a high diversity in the pathogenic potential of isolates of C. gattii belonging to the molecular types VGIII and VGIV.

Four monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, C, Y and W135: its application in identity tests.

Murine hybridoma monoclonal antibodies (MAbs) were produced against the capsular polysaccharide (CPs) of serogroups A, C, W135 and Y meningococci (MenA, MenC, MenW, MenY) in order to develop immunological reagents for the identification of meningococcal polysaccharides. Each serogroup-specific MAb reacted with the CPs from its homologous serogroup only and did not react with CPs from the other three serogroups. The affinity constant (Ka) of the four MAbs measured by non-competitive ELISA was 6.62 × 10(9), 2.76 × 10(9), 1.48 × 10(9) and 3.8 × 10(9) M(-1) for MenA, MenC, MenW and MenY MAbs respectively. The application of these MAbs for identity tests was demonstrated by their abilities to correctly identify the CPs from serogroups A, C, W135 and Y in meningococcal CPs-based vaccines through ELISA. The MAbs obtained in this work are a very valuable set of tools for study meningococcal polysaccharides vaccines.

M-protein gene-type distribution and hyaluronic acid capsule in group A Streptococcus clinical isolates in Chile: association of emm gene markers with csrR alleles.

Streptococcus pyogenes causes a variety of infections because of virulence factors such as capsular hyaluronic acid and M protein. The aim of this study was to determine emm types and capsule phenotype in 110 isolates of S. pyogenes from patients with invasive (sterile sites) and non-invasive (mainly pharyngitis) infections in Chile, and the relationship between both virulence factors. The most abundant types found were emm12, emm1, emm4 and emm28 and their distribution was similar to that seen in Latin America and developed countries, but very different from that in Asia and Pacific Island countries. Ten of 16 emm types identified in pharyngeal isolates were found in sterile-site isolates, and three of nine emm types of sterile-site isolates occurred in pharyngeal isolates; three emm subtypes were novel. The amount of hyaluronic acid was significantly higher in sterile-site isolates but did not differ substantially among emm types. Only three isolates were markedly capsulate and two of them had mutations in the csrR gene that codes for a repressor of capsule synthesis genes. We found a non-random association between emm types and csrR gene alleles suggesting that horizontal gene transfer is not freely occurring in the population.

Features of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14.

The effect of several cultivation conditions on the kinetics of bacterial growth and polysaccharide production of Streptococcus pneumoniae serotype 14 was studied. The presence in the supernatant of serotype-specific CPS (capsular polysaccharide) during growth was followed by size-exclusion HPLC and, in parallel, confirmed by using a specific latex reagent. The agitation level did not affect the production behaviour, whereas pH maintenance above 6 strongly enhanced both growth and CPS production throughout the cultivation period in flasks. Production of high-molecular-mass polysaccharide was found to be maximal between 5 and 6 h of cultivation, at the end of the exponential phase. By laser light scattering, 90% of this purified CPS product showed a M(w) (molecular mass) range from 350 to 1500 kDa, with an average M(w) of 921 kDa. Extending the culture to 24 h gave rise to a clear shift of the M(w) distribution of the polysaccharide to values lower than 100 kDa. These findings may have strong implications for the large-scale manufacture of the polysaccharide and the associated conjugate vaccine.

Salicylic acid diminishes Staphylococcus aureus capsular polysaccharide type 5 expression.

Capsular polysaccharides (CP) of serotypes 5 (CP5) and 8 (CP8) are major Staphylococcus aureus virulence factors. Previous studies have shown that salicylic acid (SAL), the main aspirin metabolite, affects the expression of certain bacterial virulence factors. In the present study, we found that S. aureus strain Reynolds (CP5) cultured with SAL was internalized by MAC-T cells in larger numbers than strain Reynolds organisms not exposed to SAL. Furthermore, the internalization of the isogenic nonencapsulated Reynolds strain into MAC-T cells was not significantly affected by preexposure to SAL. Pretreatment of S. aureus strain Newman with SAL also enhanced internalization into MAC-T cells compared with that of untreated control strains. Using strain Newman organisms, we evaluated the activity of the major cap5 promoter, which was significantly decreased upon preexposure to SAL. Diminished transcription of mgrA and upregulation of the saeRS transcript, both global regulators of CP expression, were found in S. aureus cultured in the presence of SAL, as ascertained by real-time PCR analysis. In addition, CP5 production by S. aureus Newman was also decreased by treatment with SAL. Collectively, our data demonstrate that exposure of encapsulated S. aureus strains to low concentrations of SAL reduced CP production, thus unmasking surface adhesins and leading to an increased capacity of staphylococci to invade epithelial cells. The high capacity of internalization of the encapsulated S. aureus strains induced by SAL pretreatment may contribute to the persistence of bacteria in certain hosts.

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirão Preto, São Paulo, Brazil.

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeirão Preto, São Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors.

Pneumococcal whole-cell vaccine: optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium.

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kanR, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.

Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice.

Staphylococcus aureus capsular polysaccharides (CP) have been shown to enhance staphylococcal virulence in numerous animal models of infection. Although serotype 5 CP (CP5) and CP8 predominate among S. aureus isolates from humans, most staphylococcal isolates from bovines with mastitis in Argentina are capsule negative. This study was designed to evaluate the effects of CP5 and CP8 expression on the pathogenesis of experimental murine mastitis. Lactating mice were challenged by the intramammary route with one of three isogenic S. aureus strains producing CP5, CP8, or no capsule. Significantly greater numbers of acapsular mutant cells were recovered from the infected glands 12 days after bacterial challenge compared with the encapsulated strains. Histopathological analyses revealed greater polymorphonuclear and mononuclear leukocyte infiltration and congestion in the mammary glands of mice infected with the encapsulated strains compared with the acapsular mutant, and the serotype 5 strain elicited more inflammation than the serotype 8 strain. In vitro experiments revealed that the acapsular S. aureus strain was internalized by MAC-T bovine epithelial cells in significantly greater numbers than the CP5- or CP8-producing strain. Taken together, the results suggest that S. aureus lacking a capsule was able to persist in the murine mammary gland, whereas encapsulated strains elicited more inflammation and were eliminated faster. Loss of CP5 or CP8 expression may enhance the persistence of staphylococci in the mammary glands of chronically infected hosts.

Extended phenotype of an mreB-like mutant in Azospirillum brasilense.

Tn5 mutagenesis was used to generate an Azospirillum brasilense SPF94 mutant. Genetic analysis of this mutant revealed that a homologue of the mreB gene, which controls cell shape in Bacillus subtilis and Escherichia coli, was inactivated. The cell-surface properties of the mutant were different from those of the parental strain. The mutant colonies were highly fluorescent when grown on plates containing Calcofluor White. Light and electron microscopy revealed that the mutant cells were round and had thicker capsules than the spiral parental strain. The mutants contained up to ten times more capsule protein than the parental strain, but lacked a 40 kDa protein that is abundant in the parental strain. The phenotype of the isolated mutant resembled that of the cyst-like differentiated forms of Azospirillum, suggesting that the mreB homologue could be involved in differentiation.

[Effect of cultivation parameters on the composition of extracellular polysaccharide containing substances in bacterium Azospirillum brasilense]. / Issledovanie vliianiia uslovii vyrashchivaniia bakterii Azospirillum brasilense na sostav vnekletochnykh polisakharidsoderzhashchikh materialov.

Maintenance of pH 7.0 during the fermentation period favors accumulation of high-molecular polysaccharide-containing components, the so-called lipopolysaccharide-protein and polysaccharide-lipid complexes, in the capsules and culture medium. Increased pH of the culture medium to 8.0 reduced the period of exponential growth and the yield of polysaccharide-containing complexes as compared to the optimal conditions. Maintenance of pH 5.5 suppressed the culture growth and polysaccharide production. The polysaccharide-lipid complexes obtained when pH was stabilized at the level of 7.0-8.0 had a relatively low molecular weight and contained only acidic polysaccharides. The use of potassium gluconate instead of sodium malate as a source of carbon in the culture medium changed the polysaccharide composition and increased the content of glucosamine, which increased the attraction of polysaccharides to wheat germ agglutinin. Prolongation of Azospirillum cultivation to five days introduced new glucose-containing polysaccharide components in the capsule.

Lactobacilli express cell surface proteins which mediate binding of immobilized collagen and fibronectin.

Binding of immobilized collagen-I (Cn-I) and fibronectin (Fn) by Lactobacillus acidophilus CRL 639 depends on cell-surface proteins. Capsule formation during the stationary growth phase has a negative effect on adherence of Cn-I and Fn. However, cells from the exponential growth phase, which produce no capsule, exhibit maximal binding. Binding is sensitive to trypsin, proteinase K, pronase E, and heat. Gelatin and soluble Cn-I partially inhibit binding of Cn-I although various proteins, sugars and amino acids do not affect binding to Fn. These results indicate that protein-protein interactions mediate adhesion to extracellular matrix proteins. SDS-PAGE and Western blot analyses of surface proteins revealed that several proteins including the major 43-kDa protein of the S-layer are expressed. Monoclonal antibodies showed that Fn binds to a 15-kDa protein, while Cn-I binds to proteins of 45 and 58 kDa.

The polar polysaccharide capsule of Hyphomonas adhaerens MHS-3 has a strong affinity for gold.

Select groups of bacteria, including prothescate species, have an unusual capacity to sequester gold and bioconcentrate it to very high levels. Hyphomonas adhaerens MHS-3 (MHS-3) is one such species, as demonstrated by Energy Dispersive Spectroscopy. Transmission electron microscopy revealed that the binding site was specific on the polar polysaccharide capsule. A capsuleless mutant and periodate-treated wild type did not sequester gold. The gold may interact with the same sites in the capsule that naturally adhere MHS-3 to surfaces in the marine environment.