Adherence to laminin of invasive and non‐invasive GAS isolates.
Streptococcus pyogenes (group A streptococcus – GAS) can cause numerous human infections, varying from mild skin infections to life‐threatening, e.g. necrotizing fasciitis. Adherence and biofilm production are important in streptoccocal pathogenesis. GAS adhesins are numerous and diverse, with the ability to bind to several different receptors at the same time, which leads to difficulties in their precise identification and classification. Biofilm production is one of the most probable explanation for therapeutic failure in the treatment of GAS infections. Most researchers agreed that biofilm formation is a trait of individual strains rather than a general serotype attribute. The aim of our study is to investigate differences in adherence to laminin and biofilm production between invasive and non‐invasive isolates (NI) of GAS. In this study the correlation between adherence to laminin and invasiveness in GAS isolates is noticed. The strains isolated from GAS carriers and highly invasive (HI) GAS strains have excellent capacity for binding to laminin. When testing biofilm production, there was noticeable positive correlation between adherence and biofilm production among non‐invasive isolates. Non‐invasive isolates were stable biofilm productors. There was no correlation between adherence and biofilm production among invasive isolates. Invasive isolates were also unstable biofilm productors.
- Streptococcus pyogenes
- biofilm production
On the other hand, such invasive bacteria could become normal flora‐like bacteria during pharyngeal or nasal carriage. Streptococcal carriage has been defined as the recovery of GAS from the nasopharynx or oropharynx in the absence of any evidence of acute infection . Streptococcal carriers should not be treated with antibiotics, except in the cases of reappearance of disease or possible occurence of post‐streptococcal sequelae. Genesis of streptococcal carriage was for a long time poorly understood. Nowadays, there are two theories which explain streptococcal carriage as the consequence of therapeutic failure happened after infection of strains capable to produce biofilm  or internalize into epithelial cells .
2. Adherence and biofilm production of
2.1. New insight into old problem of group A streptococcal adherence
Although sometimes not sufficiently emphasized, efficient adherence is the prime step in the pathogenesis of infective disease. Factors that influence adherence are diverse and can originate from environment such as the substrate on which the biofilm creates, initial bacterial layer coating the substrate, and characteristics of bacteria multiplying in this medium .
Adherence is complex process that includes several different steps. In the first step, bacteria have to overcome the repulsive forces which are consequence of negative charge of the bacterial superficial adhesins and substrate. Afterwards, in the second step positively attracting forces (such as covalent, ionic, van der Waals, hydrophobic) are established between bacterial adhesin and compatible receptor on the human cell. These attractive forces act on the small distance and only after bacterial surpassing of the repulsive electrostatic forces. Most of these interactions are low affinity bonds, but acting together they turn out to be strong and high affinity. Van der Waals forces play crucial role in protein‐protein recognition, when complementary lock‐and‐key shapes are involved . Hydrophobic side chains on the proteins could be connected to each other also using the low affinity hydrophobic forces. This is very plain and simple observation of adherence, and we should highlighted here that in the same or similar environmental conditions even closely related species in genus
Nowadays, there is proposition of two‐step adherence of
Bacterial adherence to human cells could be on the direct or indirect way. Direct way of adherence is displayed by binding of bacterial adhesion to specific receptor on the cell surface; e.g. capsular hyaluronic acid interacts with CD44 receptor on the surface of keratinocytes and induces reorganization of cytoskeletal actin and rupture of intercellular bridges enabling bacteria to penetrate the epithelium still staying extracellular and reaching deeper into the tisssue . The other, indirect way of adherence is more common. Streptococcal adhesins first bind to proteins of extracellular matrix (ECM) such as fibrinogen, fibronectin, laminin, collagen as bridging molecules which than attach to cell membranes integrins .
2.1.1. Fibronectin binding proteins
Fibronectin (Fn) is a high‐molecular weight glycoprotein that circulates free as a dimer in the soluble form in blood plasma or as a fibrillar form is assembled by cells as major component of the ECM. So far, fibronectin binding proteins are the best studied adhesins of
Expression of Fn‐binding proteins is regulated as response to the environmental conditions in which streptococci survive and multiply. Protein F/SfbI, which allows binding to epithelial cells of the dermis and Langerhans cells, show increased expression on bacterial surface with increasing pressure of oxygen, e.g. on the cell surface, thereby enabling a better adherence of the bacteria. When oxygen level is decreased, e.g. in deep tissue, expression of this protein is also diminished, allowing bacterial dissemination into deeper tissues . SfbI expression could be diminished also by catalytic cleavage with serine protease streptococcal pyrogenic exotoxin B (SpeB) or by other bacterial surface proteases where infection occurs. Protein F2, detected in most SfbI negative‐ GAS strains, binds fibronectin with high affinity and is homologous to Fn‐binding proteins of group C streptococci. Similarly as in protein F1/SfbI, F2 activity is also response to the environmental oxygen pressure . Unlike these two proteins, M protein expression is enhanced in the deeper tissues with increased pressure of carbon dioxide, preventing phagocytosis and contributing to the dissemination of GAS .
2.1.2. Anchorless adhesins
Anchorless adhesins are attached to bacterial cell surface in the unknown mode, probably through hydrophobic interactions. Importance of these proteins is in their ability to separate from the cell surface, get away from the cell, detects environmental signals around streptococci and the information transferred back to
2.1.3. Laminin binding adhesins
Laminin is high‐molecular weight ECM protein and one of the major components of the basal lamina, which is part of the basement membrane in human cells. Although laminin is widely distributed in our body, only a few laminin binding proteins are identified in GAS so far. Currently, proteins nominated as streptococcal hemoprotein receptor (Shr), laminin binding protein (Lbp), and streptococcal pyrogenic exotoxin B (SpeB) are identified as laminin binding proteins for
SpeB is anchorless adhesin, with enzymatic function as cysteine protease. SpeB was first identified as exotoxin, but this protein can be attached to the bacterial surface as adhesin. SpeB is synthesized during early stationary phase in nutritious poor media . This protein, like M protein, has multiple functions (adhesin, proinflammatory effect, and enzymatic function). Besides his function as laminin binding adhesin, this protein can bind to fibronectin and vitronectin, allowing streptococcal dissemination in deep tissue  and activates metalloproteases included in remodeling and degrading of ECM .
Shr is probably the protein attached to cell membrane, because it contains nor LPXTG either QVPTG repeats, that recognize housekeeping or accessory sortases, enzymes which incorporate proteins in the cell wall . Its membrane position corresponds to the primary role of Shr protein in uptaking of the heme and binding to its transporter in the cytoplasmic membrane [19, 20]. In addition to its metabolic role, and by the virtue of surface position, it has been shown that this protein have the ability to bind laminin and fibronectin, participating in this way in adherence and acting as MSCRAMMS .
Lbp belongs to the group of metal‐binding receptors with modified accessory proteins. Lbp scavenges environmental zinc and transports it to carriers of the cell membrane, to which it is attached . Also, Lbp is laminin binding adhesin . This protein is not identified in the oral streptococci, but is present in all surveyed so far M serotypes of GAS . Lbp is very short, even shorter than the thickness of the cell wall, and because of its location in the cell membrane, it is likely to have greater importance in the metabolism of metal than in the adherence to laminin .
2.1.4. Our experience with group A streptococcal adherence
Considering that the adherence of streptococci is still insufficiently examined process and that the streptococcal adhesins are numerous and irregularly and inadequately identified, in our study isolates were divided in three groups according to invasiveness of the disease they caused. The aim of our study was to investigate differences in adherence to laminin between invasive and non‐invasive isolates (NI) of GAS.
220.127.116.11. Material and methods
18.104.22.168.1. Bacterial strains
In total, 172 GAS isolates were included in the study. They were divided into three groups: (1) 100 non‐invasive isolates (NI) obtained from GAS carriers; (2) 50 low invasive (LI) isolates obtained from patients with tonsillopharyngitis; and (3) 22 highly invasive (HI) recovered from blood of patients with sepsis and STSS. All the isolates are part of the national collection of GAS strains formed at the National Reference Laboratory for Streptococci, Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade. The NI and LI isolates were collected during 2012, while HI isolates had been collected over the last two decades.
22.214.171.124.2. Laminin coating of microtiter plates
We investigated adherence of GAS strains to uncoated and laminin‐coated microtiter plates. Laminin coating of the polystyrene microtiter plates (Kartell, Italy) was performed by using 0.5 mg/ml laminin (Sigma Aldrich, USA), in accordance with the manufacturer's instructions. The plates were coated for 2 hours at 37°C with laminin previously diluted in Hanks balanced salt solution (Sigma Aldrich, USA) to achieve final concentration of 5 μg/ml, and afterwards were washed three times with Hanks balanced salt solution.
126.96.36.199.3. Capsule removal by hyaluronidase
Prior to adherence testing, all isolates were treated with bovine testicular hyaluronidase, type VI‐S, (Sigma‐Aldrich, USA) diluted in enzyme diluent (20 mM Sodium Phosphate, 77 mM Sodium Chloride, 0.01% Bovine Albumin, pH 7.0 at 37°C) in order to remove their capsules, as previously described .
188.8.131.52.4. Quantification of adherence to laminin by GAS strains
Quantification of adherence of GAS strains to uncoated and laminin‐coated microtiter plates was based upon the protocol described by Stepanovic
OD ≤ ODc = non‐adherent isolates, ODc < OD ≤ (2 × ODc) = weakly adherent isolates (+), (2 × ODc) < OD ≤ (4 × ODc) = moderately adherent isolates (++) and OD > (4 × ODc) = strongly adherent isolates (+++). All analyses were performed in triplicate and repeated at least two times.
184.108.40.206.5. Statistical analysis
220.127.116.11. Results and discussion
To determine correlation between invasiveness of tested GAS strains and their ability to bind to laminin, we investigated adherence of NI, LI, and HI isolates to uncoated and laminin‐coated microtiter plates. All isolates were treated with hyaluronidase in order to eliminate the interference of hyaluronic acid capsule on adherence. The proportions of NI, LI, and HI isolates that displayed adherence to uncoated microtiter plates were 98%, 71%, and 91%, respectively. In all adherent isolates the level of adherence was estimated as weak, but adherence of NI and HI isolates to uncoated plates was significantly higher than adherence displayed by isolates of the LI group (
|No (%) of weakly adherent|
|No (%) of moderately|
|No (%) of strongly|
|NI group (total 100 isolates)||13 (13)||40 (40)||47 (47)|
|LI group (total 50 isolates)||13 (26)||27 (54)||10 (20)|
|HI group (total 22 isolates)||0||5 (23)||17 (77)|
The overall results showed significantly higher adherence (
In conclusion, this study showed correlation between adherence to laminin and invasiveness in GAS isolates. The strains isolated from GAS carriers and highly invasive GAS strains have excellent capacity for binding to laminin.div4close
The greatest importance of the effective bacterial adherence is in the attachment to host cells and the aggregation of bacteria, which then create a signal for the biofilm production. In a collective way of existence bacteria gain a protective matrix layer, which in planktonic lifestyle does not exist and is responsible for the most of mechanisms that bacteria avoid eradication from the infection site.
According to literature,
Biofilm production is one of the most probable explanation for therapeutic failure in the treatment of infections with this bacteria,
Fibronectin‐collagen‐T antigen (FCT) classification is only partially managed to link biofilm production with certain FCT groups. In FCT region of
2.2.1. Our experience with biofilm production of
Considering that biofilm production, like adherence, is still not sufficiently explained virulence factor, we supposed that dividing isolates according to invasiveness would be interested. Like in adherence experiments, we also divided isolates in three groups in order to show correlation between biofilm production and invasiveness of strains tested.
Our goal was to find out whether biofilm production as virulence factor is correlated with specific disease/clinical condition. Considering that adherence and hydrophobicity are in relationship with biofilm production, we also wanted to show possible association between them.
2.2.2. Material and methods
18.104.22.168. Determination of hydrophobicity
Hydrophobicity was measured by two different methods described previously by Rosenberg
22.214.171.124. Biofilm production
Biofilm production was determined by the same methodology  as adherence testing with modification in incubation period of 12, 24, and 48 hours. As in adherence testing, according to ODc isolates were designed as non‐producers = OD < ODc, weak biofilm producers = ODc < OD ≤ (2 × ODc) (+), (2 × ODc) < OD ≤ (4 × ODc) = moderate biofilm producers (++) and OD > (4 × ODc) = strong biofilm producers (+++).
126.96.36.199. Statistical analysis
ANOVA was used to determine the differences in hydrophobicity and biofilm production among different groups of GAS strains. Correlation between adherence, hydrophobicity, and biofilm production was determined by Pearson test. Data analyses were done with the SPSS version 20. The differences were considered significant if
Adherence results are shown in Section 188.8.131.52.
Measurement of bacterial hydrophobicity was first performed by MATH test using hexadecane as hydrocarbon after removal of the capsule, which hinders superficial hydrophobic proteins. Adherence to hexadecane was very low and with no statistical difference between groups. After that we tested GAS adherence to xylene. In our assay adherence to xylene were 48.49, 22.78, and 36.09 for NI, LI, and HI group, respectively. It was noticed statistically significant difference between groups, particularly NI group isolates were more hydrophobic in relation to other two groups (
184.108.40.206. Biofilm production
When we did dynamic analysis of biofilm production during specified incubation periods (12–48 hours) all three groups have shown different pattern (
220.127.116.11. Analysis of correlation between adherence, hydrophobicity and biofilm production
Adherence and hydrophobicity are very important in process of the biofilm formation. We used various measurement methods to establish possible connection between these three traits of bacteria.
When non‐invasive group of isolates was analyzed, positive correlations were noticed between adherence and biofilm formation after 48 hours of incubation (
When low invasive group of strains was explored, no correlation was noticed between adherence, hydrophobicity, and biofilm production (
When highly invasive group of isolates was studied, no correlation was noticed between adherence, hydrophobicity, and biofilm production (
In this study it is demonstrated correlation between adherence, hydrophobicity and biofilm production for non‐invasive isolates, while for low and highly invasive isolates no correlation was noticed.
Group A streptococcal adherence is still unrevealed process depending on unspecific hydrophobic bonds and on specific protein‐protein or protein‐carbohydrate interactions. Hydrophobic interactions are weak non‐covalent interactions between water and hydrophobe (non‐polar low‐water soluble molecules). Hydrophobic interactions are stronger than other weak intermolecular forces (van der Waals or Hydrogen bonds) and depend on several factors: temperature, number of carbon atoms on hydrophobe and shape of hydrophobe.
According to Rosenberg
Non‐invasive isolates from streptococcal carriers have shown direct, positive relationship between adherence to uncoated microtiter plate and late stage biofilm production. These isolates were also the most stable biofilm producers during all three incubation intervals, confirming the latest theory that biofilm production could be possible explanation for pharyngeal carriage . Marks
In our study, we did not find any relationship between different methods of adherence and hydrophobicity measurements for low and highly invasive isolates. Also, we showed that highly invasive isolates have been unstable biofilm producers, contributing to previous findings of other researchers that biofilm production is not crucial virulence factor for invasive strains.
According to literature, this was first work about determination of the relationship between adherence, hydrophobicity and biofilm production for
In conclusion, it is obvious that adherence and biofilm production are not phenotypic traits of all species, but rather individual characteristic of every strain. It is important to emphasize that our experiments have been conducted
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