report

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Host protection mechanisms against
Group A Streptococcus in human
whole blood
Molecular and Cellular Life Sciences
By: Pier Paolo Posata
Supervision: Nina van Sorge, Suzan Rooijakkers
Daily supervision: Nina van Sorge

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Laymen`s summary
Group A Streptococcus (GAS) is a Gram-positive bacterium that has no reservoir outside the
human host. Although most of GAS infections are mild, it is also able to cause also life-
threatening diseases which lead to over 500,000 deaths worldwide each year; therefore, GAS
is considered a dangerous threat for humans. The reason that why some GAS strains gain
access to deeper tissues in human body, leading to life-threatening diseases, is still unknown,
but it is probably related to its survival in human blood and its evasion from innate immune
system. It is well-established that GAS growth in whole blood is promoted by virulence
factors. Nevertheless, these virulence factors are not able to promote growth in all blood
donors. In our research, we investigate host protection mechanisms against GAS in human
whole blood. We demonstrate that the same GAS strain causes different phenotypes according
to the donor.

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Table of Contents
Introduction ................................................................................................................................ 4
Phagocytosis ........................................................................................................................... 4
Group A streptococcus ........................................................................................................... 5
GAS survival in human blood ................................................................................................ 5
Research aim .............................................................................................................................. 6
Results ........................................................................................................................................ 7
Donor differences in whole blood killing assay ..................................................................... 7
Reproducibility of whole blood killing assay......................................................................... 8
GAS grows similar in plasma from different donors.............................................................. 9
Importance of neutrophils numbers and phagocytosis for the whole blood phenotype ....... 10
Opsonic capacity of individual sera...................................................................................... 11
Neutrophil killing capacity ................................................................................................... 14
Donor differences in neutrophils killing capacity ................................................................ 15
Discussion ................................................................................................................................ 16
Materials and methods ............................................................................................................. 19
References ................................................................................................................................ 21

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Introduction
Phagocytosis
Phagocytosis is a biological process through which cells engulf and digest particles. It plays
an essential role in the human innate immunity, which is designed to eliminate invading
pathogens. A specialized group of cells, collectively called phagocytes, are involved in killing
bacteria through phagocytosis. These professional phagocytes include monocytes,
macrophages, neutrophils, mast cells and dendritic cells which are all able to phagocytose
with high efficiency.
This research focuses on neutrophils. Neutrophils are the most important phagocytic cells of
the innate immune system and they represent around 60% of the white blood cells. They are
“killing machines” that defend the host against infections, by internalizing the pathogens and
killing them with reactive oxygen species (ROS), proteases, antimicrobial peptides or other
enzymes (Beutler, 2004).
For efficient neutrophil phagocytosis, it is required that bacteria are covered with specific
molecules, called opsonins. Opsonins label the target pathogen, which must be internalized,
and promote phagocytosis. Antibodies (immunoglobulins (Ig)) and complement proteins are
the two dominant and well-studied opsonins, but other innate immune components might act
as opsonins as well.
Immunoglobulins are Y-shaped glycoproteins highly abundant in blood and they are produced
by specific cells, called B cells. An immunoglobulin identifies a unique part of the foreign
target, called an antigen (Janeway, 2001). The variable region determines the specificity of
the Ig and it differs for each epitope. Immunoglobulins exist in different isotypes, which have
different sizes and composition. Humans have five different isotypes: IgG, IgA, IgM, IgE and
IgD (Burton & Woof, 1992). This research focuses on IgG, the
main Ig present in plasma and extracellular fluids.
In addition to Ig, pathogens can be labeled for phagocytosis by
the complement system. The human complement system
consists of a number of small proteins, which are activated by
cleavage. The complement system is triggered in three different
ways (Ig, carbohydrates and spontaneous), and all result in C3
cleavage resulting in C3b deposition on the target particle
facilitating phagocytosis (Vidarsson, 1998). Furthermore, some
of the released factors attract neutrophils towards the infections
in a process called chemotaxis (Ward, 1965).
Once the opsonization occurs, phagocytes recognize the
opsonized bacteria by means of specific receptors on their
surface, called IgG receptor (FcR) and complement receptors
(CR) (Vidarsson, 1998) (Fig. 1). FcRs are membrane
glycoproteins, which are classified based on the type of
immunoglobulin that they recognize (Heijnen, 1997). These
receptors recognize the constant part of Fc domain of Ig and
initiate internalization. On the other hand, the complement
receptors recognize fragment of complement proteins that have
been hydrolysed upon opsonization (Holers, 2014).
Subsequently, phagocytes take up pathogens in vesicles, called
phagosomes, and fuse them with specific granules, which
contain antimicrobial molecules, like elastase, azurocidin,
cathepsin G, LL-37, to kill the bacteria (Turner, 1998).
Figure 1| Phagocytosis of
bacteria by neutrophils
through opsonins.
Mechanism by which
neutrophils internalize
bacteria. Bacteria are
opsonized by complement
proteins and antibodies
which are recognized by
neutrophil though CR
receptors and FcR
receptors, respectively.
Subsequently, pathogen is
taken up in vesicles that
fuse with specific granules.

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Furthermore, neutrophil activation lead to the release of reactive oxygen species (ROS) which
damage bacterial nucleic acids, proteins and cell membranes through a process called
oxidative burst (Hampton, 1998).
In summary, neutrophils are fully equipped to kill Gram-positive bacteria and prevent
infections, nevertheless Gram-positive bacteria are not a harmless victim of the immune
system mechanisms.
Group A Streptococcus
Group A Streptococcus (GAS) is a spherical Gram-positive bacterium that causes infections
in humans. GAS is a human restricted pathogen responsible for infecting over 700 million
people in the world each year (Carapetis, 2005). Most of these infections are mild like
pharyngitis and impetigo which affect respiratory tract and superficial skin, and they are cured
easily with antibiotics. On the other hand, GAS strains may colonize deeper tissues, the blood
stream, lymphatics and various organs, causing life-threatening diseases like necrotizing
fasciitis and toxic shock syndrome. The result is 500,000 deaths worldwide each year
(Carapetis, 2005; Cunningham, 2000).
GAS survival in human blood
The fact that GAS causes such high mortality and morbidity suggests that it is able to resist
detection and killing by the human immune system. It is thought that the ability of clinical
isolates of GAS to survive in human blood is an important virulence determinant (Lancefield,
1957). Thus far, several virulence factors have been identified that promote survival of GAS
in human whole blood by interfering with opsonization and phagocytosis. For example, the
cell wall-associated M protein is a well-defined virulence factor and also underlies the
classification of GAS into serological groups: over 200 emm genotypes have been
characterized (McMillan, 2013). M protein confers an high pathogenicity on GAS and most of
the GAS virulence is attributed by M protein due to different mechanisms. Firstly, it has a
weak immunogenicity due to its antigenic variation which allows to escape antibody attack
(Lannergard, 2011). Secondly, it recruits fibrinogen and other plasma proteins, blocking the
complement pathway and inhibiting C3b deposition (Horstmann 1992; Carlsson, 2005;
Fischetti, 1989). In addition, GAS is also able to inhibit neutrophils chemotaxis through a
molecule known as C5a peptidase. C5a peptidase cleaves complement peptide C5a, which
binds its neutrophil complement receptor (C5aR) and recruits neutrophils towards the
infection (Wexler, 1985; Joiner, 1983). Besides M protein and C5a peptidase, GAS has other
important virulence factors that interfere with opsonization and phagocytosis: the capsule and
the cell wall are considered to play an important role in GAS survival. The capsule is
composed of hyaluronic acid, and it surrounds and protects the bacterium from phagocytosis,
increasing GAS virulence (Moses, 1997). In fact, hyaluronic acid obstruct antibody binding to
epitopes on the bacterial surface, and complement deposition (Dale, 1996). The cell wall is
composed of peptidoglycan and it has important antigens, like the Lancefield group A
carbohydrate (GAC) which constitutes the 50% of GAS cell wall by weight (McCarty, 1952)
and has been recently discovered to enhance GAS pathogenesis (van Sorge, 2014).
In addition to interference with phagocytosis and chemotaxis, GAS is also able to survive
inside human neutrophils (Cunningham, 2000; Staali, 2003) through different mechanisms.
First, it has been described that M protein prevents the fusion of azurophilic granules with
phagosomes and this would protect GAS from the action of neutrophils antimicrobial
molecules (Staali, 2006). Second, once internalized by neutrophils, M protein may also
support GAS to resist antimicrobial peptides killing (Lauth, 2011). Third, GAS exploits

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different factors for oxidative stress resistance: the thickness of the capsule reduces oxygen
absorption shielding GAS from ROS (Cleary & Larkin, 1979); several GAS surface proteins
regulate cation homeostasis enhancing ROS resistance (Henningham, 2015).
In contrast to the many virulence factors that promote GAS survival, much less is known
about the host factors that contribute to GAS killing and control infection.
Research aim
Previous experiments by van Sorge at the University of California San Diego, identified that
there are inter-individual differences with respect to GAS blood survival, which are not
related to strain differences (Fig. 2). For this reason, virulence factors are not able to promote
survival in all blood donors suggesting differences in host defence mechanisms.
The aim of this research is to identify host protection mechanisms against GAS in
human whole blood.
We know from figure 2 that GAS survival depends on the donor: when using the same GAS
strain, some donors kill GAS, and others support GAS growth. Our goal is to test whether
these donor differences are observed and reproducible in Utrecht. Furthermore, the outcome
of these experiments will provide an opportunity to investigate the importance of host
protection mechanisms against GAS infection, like phagocytosis, complement, IgG levels and
neutrophils killing capacity, by comparing different phenotypes between donors.
Whole blood killing
0 1 2 3
0.1
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Time (h)
Survival
(%,comparedtot0)
Jason (5/10/11)
Kathryn (22/10/10)
Cheryl (24/2/11)
Markus (18/8/11)
Laura Crotty (23/8/11)
Andrew (21/9/11)
Josh (3/10/11)
Kathryn (27/9/11)
Josh (22/9/11)
Markus (26/9/11)
Figure 2| Different
phenotypes in whole
blood killing assay as
determined by van
Sorge. The graph shows
survival of GAS in 10
different donors (each
line is a donor). 100%
survival represent a
steady-state between
time=0 and the other
time points. Donors
have different
phenotypes. Most of
them support GAS
growth but two kill the
bacteria after 3 hours
(red lines).

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Results
Donor differences in whole blood killing assay
Previously, van Sorge identified that the same GAS strain could either be killed or
demonstrate growth when incubated with blood from different donors. To test whether these
donor differences were also present in Utrecht, we selected six different donors. The same
GAS strain was incubated with blood from six donors and survival was measured after three
hours incubation. Similar to observations at UCSD, three different phenotypes were observed.
Donors 1 and 4 kill GAS: “Killers” (red bars), donors 2 and 5 support GAS growth:
“Growers” (green bars) and donors 3 and 6 contain GAS at steady-state levels: “Containing
donors” (blue bars) (Fig.3). According to these results, changing donors on GAS tests would
alter the outcome of the results. Therefore, not only GAS strains employed but also the type
of donor is important for GAS growth in blood.
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Figure 3| Three different phenotypes in whole blood killing assay in Utrecht. The bar graph shows the
survival of GAS in blood from six different individuals after 3 hours incubation compared to number of
colonies at time=0. The dotted line represent a steady-state between time=0 hours and time=3 hours. Three
different phenotypes emerge and they are depicted in three colors (red, green and blue), which will represent
the three phenotypes throughout this paper. Donors who kill the bacteria are red, donors who support GAS
growth are green and donors in which GAS growth remains steady are blue. The experiment was performed in
quadruplicates and the error bars represent the standard deviation of technical replicates.
Time 3 hours

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Reproducibility of whole blood killing assay
Once we confirmed the three different phenotypes in different donors, we investigated
whether the phenotype was reproducible. Blood contains a copious number of factors, which
may not be stable from day to day and therefore influence the outcome of the experiment. For
this reason, we tested donor 1-4 again 1.5 months later (27/11/2014) with the exact same
assay we performed previously. Results demonstrate that the GAS growth phenotype remains
stable in the same donor even after almost two months (Fig.4). Therefore, the donor
differences are stable and reproducible over time, This suggests that host protection
mechanisms against GAS remain constant within a donor. It is possible to conclude that the
blood donor is very important for GAS survival as well as GAS strain, and host protection
mechanisms against GAS differ from donor to donor.
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Growthindex
(comparedtoT=0)
1
D o n o r 1
D o n o r 2
D o n o r 3
D o n o r 4
Figure 4| Different GAS killing phenotypes in human blood remain constant over time. Using the same
parameters, we re-tested GAS survival in another whole blood killing assay using donors 1-4. The graph shows the
comparisons between the first whole blood killing assay 02.10.2014 (fig. 2), and the second whole blood killing
assay, 27.11.2014. The color of the bars corresponds to the whole blood killing phenotypes. The experiments were
performed in quadruplicates and the error bars represent the standard deviation of the technical replicates.
Time 3 hours

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GAS grows similar in plasma from different donors
As we established that the donor difference were reproducible, we investigated different
components of the immune system that could contribute to these different phenotypes in
whole blood. We first checked whether the whole blood phenotype could be explained by
differences in plasma survival. The plasma that we used was collected from the same donors
at the same time using lepirudin (inhibitor of thrombin) as an anticoagulant. GAS was equally
capable of growing in plasma collected from “Growers”, “Killers” or “Containing donors”
(Fig.5). These findings suggest that the acellular component of blood by itself does not
explain the outcome of the whole blood killing differences in the donors. Therefore, the whole
blood killing phenotype is probably due to the donors cells or, perhaps, a combination of
donors cells and plasma.
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Growthindex
(comparedtoT=0)
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Figure 5| Plasma survival assay. The bar graph shows the growth of GAS in plasma after 3 hours
compared to inoculum at time=0. Plasma from six donors was tested and the dotted line represent a steady-
state between time=0 and time=3 hours. PBS was used as a control GAS grows equally in all six donors.
The color of the bars corresponds to the whole blood killing phenotypes. The experiment was performed in
triplicates and the error bars represent the standard deviation of the technical replicate.

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Importance of neutrophil numbers and phagocytosis for the whole blood phenotype
To discover what underlies these donor differences, we focused on possible differences in
neutrophils numbers and neutrophil-mediated functions in the four different donors. The
number of neutrophils circulating in the blood might determine GAS killing or survival in
blood. As expected, we observed 4-fold differences in the amount of neutrophils between
donors (Fig. 6A) but this did not directly correlate with GAS survival phenotypes. For
example, donor 3 has the highest number of neutrophils but did not kill the bacteria after 3
hours. However, donor 2 had the lowest number of neutrophils (Fig. 6A) and was unable to
contain GAS. This could indicate that there is a threshold level of neutrophils required to
contain or kill GAS in whole blood. In addition to neutrophil numbers, we investigated
whether there were differences between these four donors in the phagocytic capacity of their
neutrophils in whole blood. In parallel to the whole blood killing assay, we also incubated
FITC-labeled GAS with lepirudin-anticoagulated blood from the different donors for 30
minutes. We determined the percentage of neutrophils that phagocytized GAS as well as the
level of fluorescence of the neutrophils as read out of amount of engulfed bacteria. Donor 2, a
“Grower”, employs 70% of the neutrophils to internalize GAS, more than the other donors:
donor 1, 50%, donor 3, 40%, and donor 4, 60% (Fig 6B). Neutrophils from donors 1, 3, and 4
have taken up similar levels of GAS, but the neutrophils from donor 2, internalized three to
four times more bacteria as indicated by the increase in fluorescence (Fig 6C). These results
suggest that the low number of neutrophils in donor 2 might be compensated for more
efficient phagocytosis of GAS. However, the increased phagocytosis in donor 2 is not
sufficient to inhibit GAS growth in human blood. Therefore, the number of neutrophils might
be more relevant than phagocytosis in determining whole blood killing phenotype in the
“Grower”. On the other hand, donor 3 has a high number of neutrophils and a similar whole
blood phagocytosis assay to donor 1 and 4; for this reason, neutrophils number is important to
reduce GAS growth but does not guarantee killing of GAS.
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Numberofneutrophils
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FITC-labeledneutrophilsgeommean
Figure 6| Relative number of neutrophils and whole
blood phagocytosis of GAS in different donors. (A)
Relative amount of neutrophils in donors 1,2,3, 4. (B)
The percentage of neutrophils that have internalized
GAS in the four donors. More neutrophils have engulfed
GAS in the “Grower”. (C) Level of fluorescence of the
neutrophils in the four donors. Between 3 and 4-fold
more bacteria are internalized in the “Grower”
compared to the other donors.
The color of the bars corresponds to the whole blood
killing phenotypes. The experiments were performed in
triplicates and the error bars represent the standard
deviation of the technical replicate.
A B
C

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Opsonic capacity of individual sera
In addition to neutrophil numbers, we investigated whether there were differences in the
opsonizing capacity of plasma from the different donors. We know that humoral immunity by
itself does not prevent GAS growth. We therefore investigated the importance of two
important opsonins, antibodies and complement, by determining:
1) IgG opsonization/GAS-specific IgG levels in serum.
2) Neutrophil phagocytosis induced by individual sera
3) Role of complement in neutrophil phagocytosis.
Firstly, we investigated differences in IgG opsonization of GAS between donors. IgG was
detected on the GAS surface by means of a fluorescent protein (protein G-alexa488) which is
able to recognize all human IgG subclasses. The level of fluorescence is an indication of the
level of GAS-specific IgG. As expected, the levels of IgG directed against GAS differ
between donors, with a maximum difference of 2-fold at the highest serum concentration
tested. Levels of GAS-specific IgG were lowest in serum from donor 2 (Grower), whereas the
“Containing donor`s” serum shows the highest level of GAS-specific IgG. IgG levels in
serum from “Killers” are very similar and intermediate to donor 2 and 3 (Fig. 7). This data
suggests that 2-fold difference in IgG levels does not enhance whole blood phagocytosis, and
it seems to have no correlation with the whole blood phenotypes. For this reason, other
mechanism must play a role in GAS killing.
0 .0 0 .1 0 .2 0 .3 0 .4 0 .5 0 .6 0 .7 0 .8 0 .9 1 .0
0
1 0 0 0 0
2 0 0 0 0
3 0 0 0 0
S e ru m c o n c e n tra tio n %
IgGopsonizedbacteriageommean
D o n o r 1 H I s e ru m
Figure 7| Differences in GAS-specific IgG levels between donors. The graph show the level of fluorescence
as an indication for the level of IgG antibodies directed against GAS. HI serum from donors 1-4 was used at
different concentrations. At increasing serum concentrations, more bacteria are labeled with IgG. Each donor
show different levels of opsonization, except for donors 1 and 4 which have a very similar trend. The color of
the lines corresponds to the whole blood killing phenotypes.

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Secondly, we studied the capacity of the different sera to induce phagocytosis by using
neutrophils from an independent donor to investigate whether this correlates to the whole
blood phenotype. We incubated purified neutrophils from an independent donor A with serum
from donors 1-4 (Fig. 8A) and neutrophils from donor B with plasma from donors 5 and 6
(Fig. 8B). Phagocytosis in absence of serum/plasma was minimal with either neutrophils
donor. Overall, neutrophils from donor A demonstrate high levels of phagocytosis with serum
from every donor over the entire concentration range (Fig. 8A). Using donor B neutrophils
and plasma, phagocytosis levels are much lower: the donor 5 has a reduced phacoytosis
compared to the donor 6 and they both have 2/4-fold less engulfed bacteria compared to
donors 1-4 (Fig. 8B). This suggests that the choice of neutrophils and clotting factors might
influence neutrophils phagocytosis: GAS might use clotting factors to protect from
phagocytosis (Horstmann 1992). In addition, we observed that the serum of some “Growers”
causes reduced phagocytosis. This may cause GAS survival in whole blood in some
“Growers”. Nevertheless in donor 2, serum does not induce any reduced phagocytosis
compared to the serum of a “Killer”. Therefore, opsonins from the “Growers” are not always
related to a reduced phagocytosis in a purified system.
0 5 1 0 1 5 2 0
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D o n o r 1 s e ru m
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P la s m a c o n c e n tra tio n %
D o n o r 5 p la sm a
D o n o r 6 p la sm a
Figure 8| Neutrophil phagocytosis assay.
The graph shows the level of fluorescence of the neutrophils as read out of amount of engulfed bacteria in the
six donors. (A) Serum from donors 1-4 was tested with neutrophils from a donor A. (B) Plasma from donors
5,6 was tested with neutrophils from a donor B. The color of the lines corresponds to the whole blood killing
phenotypes.
A
B

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As we noticed some differences in phagocytosis between different donors in the previous
experiment, we decided to investigate the contribution of the complement system to
phagocytosis in the different donors. We used the same neutrophils phagocytosis assay, and
tested serum and heat-inactivated (HI) serum, in which complement proteins are inactive,
from each donor (neutrophils from donor A). We observed that the fluorescence of
neutrophils, as a measurement of internalized bacteria with active and HI serum, is very
similar between the donors, except for donor 2 which has a slight decrease of phagocytosis at
10-20% of HI serum (Fig. 9). The complement system does not significantly contribute to
GAS phagocytosis, and the whole blood killing phenotype is not due to donor differences in
phagocytosis mediated by the complement system, although the “Grower” seems to exploit
the complement system for phagocytosis more than the other donors.
0 5 1 0 1 5 2 0
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D o n o r 1 se ru m
D o n o r 1 H I se ru m
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D o n o r 2 se ru m
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S e ru m co n ce n tratio n %
D onor 3 serum
D onor 3 H I serum
0 5 1 0 1 5 2 0
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1 0 0 0 0 0
1 5 0 0 0 0
2 0 0 0 0 0
2 5 0 0 0 0
D o n o r 4 se ru m
Neutrophil killing capacity
Figure 9| Effect of complement on neutrophil phagocytosis of GAS. The graphs show the level of
fluorescence of the neutrophils as read out of amount of engulfed bacteria. Different concentrations of active
serum and heat-inactivated (HI) serum were used to test neutrophil phagocytosis in the four donors. (A) Donor
1, (B) donor 2, (C) donor 3, (D) donor 4 all have the same amount of engulfed bacteria both with serum and HI
serum. The color of the lines corresponds to the whole blood killing phenotypes.
A
C
B
D

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Thus far, our data suggest that the amount of neutrophil and opsonins may contribute to
reduce GAS growth in human blood but do not seem sufficient to fully explain the donor
differences and GAS killing. Since phagocytosis does not necessarily result in bacterial
killing, we investigated whether sera/plasma samples from different donors induced
differential killing by neutrophils.
We first optimized the neutrophil killing assay to establish the best conditions to asses
neutrophil killing capacity. Bacteria were first opsonized with either 1% or 10% human
pooled serum (HPS), incubated with and without neutrophils isolated from an independent
donor C, in either round bottom plates or siliconized tubes. GAS survival was assessed at two
different time points: 30 minutes and 3 hours. After 30 minutes, there was killing in the
samples with neutrophils both in round bottom plates and siliconized tubes. But after 3 hours,
GAS killing was only observed in siliconized tubes (Fig.10). The result suggests that
neutrophils kill GAS at increasing concentrations of HPS, whereas higher concentrations of
HPS support GAS growth in the absence of neutrophils; GAS is not able to grow in RPMI
without serum (not shown). Therefore, HPS is essential to GAS to growth but it is also
important to increase the efficiency of killing in the presence of neutrophils under these
experimental conditions. We decided to test differences between donors employing
siliconized tubes and using 3 hours incubation since these conditions are similar to the whole
blood killing assay conditions.
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- n e u tro p h ils 1 0 % H P S
+ n e u tro p h ils 1 0 % H P S
- n e u tro p h ils 1 % H P S
+ n e u tro p h ils 1 % H P S
Figure 10| Optimization of GAS neutrophil killing assay. Bacteria were opsonized with either 10% or 1%
human pooled serum (HPS) and incubated with and without purified neutrophils, isolated from a donor C, for
30 minutes and 3 hours in RPMI using either round bottom plates or siliconized tubes. Blue bars represent the
control without neutrophils and red bars with neutrophils. Bars over the dotted line indicate growth of GAS,
whereas bars below 1 indicate reduced growth or killing, all compared to the number of colonies at time 0. The
experiment was performed in triplicates and the error bars represent the standard deviation of the technical
replicate.

15
Donor differences in neutrophils killing capacity
As previously shown, plasma itself supports GAS growth without any differences between
donors, but it very important for an efficient opsonisation and subsequent phagocytosis.
However, these parameters do not necessarily correlate with the endpoint, i.e. killing of GAS.
For this reason, we tested the capacity of plasma and serum from different donors to induce
killing of GAS by neutrophils. We isolated neutrophils from two independent donors D and E
and tested them in the presence of either serum or plasma from donor 1-4, respectively. We
performed this experiment both with serum, which lacks clotting factors, and with plasma,
which reflects better the whole blood situation, to investigate in addition whether clotting
factors, like fibrinogen, may influence GAS killing or growth.
Overall, serum and plasma from different donors indeed have a different capacity to induce
GAS killing by neutrophils. With serum from donor 1, 2 and 4, neutrophils are able to kill
GAS, whereas neutrophils are unable to kill GAS in the presence of serum from donor 3 (Fig.
11A). With plasma we observe that only donors 1 and 3 induce killing, whereas plasma from
donors 2 and 4 contain GAS (Fig. 11B). These results seem to suggest that plasma and serum
have a different capacity to support neutrophil killing of GAS, but results are difficult to
interpret due to the use of different neutrophil donors. (Fig. 9). Overall, these data suggest that
both serum and plasma can support neutrophils to kill GAS but the killing differs from donor
to donor.
T
=
3
D
o
n
o
r
1
s
e
ru
m
T
=
3
D
o
n
o
r
2
s
e
ru
m
T
=
3
D
o
n
o
r
3
s
e
ru
m
T
=
3
D
o
n
o
r
4
s
e
ru
m
0 .1
1
1 0
Growthindex
(comparedtoT=0)
- n e u tro p h ils 1 0 % se ru m
+ n e u tro p h ils 1 0 % se ru m
T
=
3
D
o
n
o
r
1
p
la
s
m
a
T
=
3
D
o
n
o
r
2
p
la
s
m
a
T
=
3
D
o
n
o
r
3
p
la
s
m
a
T
=
3
D
o
n
o
r
4
p
la
s
m
a
0 .1
1
1 0
Growthindex
(comparedtoT=0)
- n e u tro p h ils 1 0 % p la sm a
+ n e u tro p h ils 1 0 % p la sm a
Figure 11| Neutrophil killing
assay. (A) Bacteria were
opsonized with 10% serum and
incubated with and without
neutrophils, isolated from a donor
D, for 3 hours in RPMI using
siliconized tubes. (B) Bacteria
were opsonized with 10% plasma
and incubated with and without
neutrophils, isolated from donor
E, for 3 hours in RPMI using
siliconized tubes. Blue bars
represent the control without
neutrophils and red bars with
neutrophils. The experiments
were performed in triplicates and
the error bars represent the
standard deviation of the technical
replicate.
A
B

16
Discussion
The reason why in some individuals GAS gains access to deeper tissues in human body,
leading to life-threatening diseases, is largely unknown. GAS survival in human blood and its
evasion from innate immune system are probably the main reasons that would explain why
GAS causes invasive infections rather than mild infections. Virulence factors, especially M
protein, promote GAS survival in human blood and evasion from immune system (Le Breton,
2012; Cunningham, 2000) but this is not always the case. Employing a virulent GAS strain,
M1T1 (Kansal, 2000), we demonstrate that GAS survival in human blood is very much donor
dependent: some donors support GAS growth and others kill the bacterium. In particular, we
identify three different donors phenotypes: the “Killers”, the “Growers” and the “Containing
donors”. These donors might have different protection mechanisms, which cause their whole
blood phenotype. A donor that kills GAS might be less susceptible in contracting an invasive
infection from GAS. Therefore it is important to investigate the defence mechanisms to
contribute to GAS killing
Importantly, we show that the GAS whole blood killing phenotype is reproducible over time
in different donors. This is crucial when one aims to identify host factors that are implicated
in killing of GAS. However, it is important to consider that also other bacteria might cause the
same blood phenotypes, and therefore we need to confirm whether the blood phenotypes are
GAS-specific testing other bacteria. In an attempt to pinpoint critical factors contributing to
killing of GAS in whole blood, we investigated different components of the innate immune
system, including neutrophil numbers, neutrophil phagocytosis in whole blood and levels of
opsonisation (Table 1).
The four donors show the main differences in the neutrophils number, in GAS phagocytosis
by neutrophils in whole blood and in the level of GAS-specific IgG (Table 1). However, it is
very difficult to find a correlation between differences highlighted in table 1 and whole blood
phenotypes. Besides neutrophil number (Fig. 6A), differences in neutrophils phagocytosis
might be a cause of the whole blood phenotypes. However, the 2-fold donor differences in
GAS-specific IgG seem not to influence neutrophil phagocytosis levels in a purified system
which is high in all four donors (Table 1). This suggest that differences in quality of IgG
rather than levels likely impact GAS phagocytosis. Despite we did not investigate whether
there were differences in IgG subclasses in the GAS-specific IgG, this might be important for
the induced effector functions since IgG 1 and 3 are better at activating neutrophil FcR
(Vidarsson, 1998). Since complement contribution is minimal (Fig. 9), GAS-specific IgG
subclasses involved in activating FcR, like IgG1 and IgG3, might be more important for GAS
Donors Whole blood
phenotype
Growth
in
plasma
Neutrophils
number
Whole blood
Phagocytosis
Opsonins
contribution
in
phagocytosis
Complement
contribution
to
phagocytosis
GAS-
specific
IgG
1 “Killer” ++++ +++ + +++ + ++
2 “Grower” ++++ + ++++ +++ ++ +++
3 “Containing
donor”
++++ ++++ + +++ + ++++
4 “Killer” ++++ ++ ++ +++ + +++
Table 1| Summary of donor differences in protection mechanisms. The table represents a schematic overview of
a part of the results. The outcome of each experiment is expressed as +: + indicates very low level, ++ indicates a
low level, +++ indicates medium level and ++++ indicates high level. This helps to compare the results of each
experiment between the four donors. The main differences between donors are in neutrophils number, whole blood
phagocytosis and GAS-specific IgG.

17
phagocytosis rather than GAS-specific IgG subclasses involved in activating CR.
Furthermore, a reduced phagocytosis in donor 5, a “Grower”, (Fig. 8B) might be due to a
lower affinity of GAS-specific IgG subclasses towards M protein which exploits its antigenic
variation to escape antibody attack (Lannergard, 2011), but we would require donor
immunoprofiling to investigate this.
In addition to host protection mechanisms shown in table 1, we know that GAS is able to
survive inside neutrophils (Staali, 2006), therefore the killing capacity of neutrophils is very
important. As a consequence, phagocytosis might not correlate directly with neutrophil killing
and it might differ by the induction of serum/plasma of different donors. For this reason, we
investigated the capacity of plasma and serum to induce neutrophil killing. Firstly, the results
show that neutrophils are able to kill GAS in the presence of HPS only in siliconized tubes
after 3 hours (Fig.10). This occurs probably because silicone avoids the activated neutrophils
to adhere to the tube unlike round bottom plates, in which neutrophils adhere to the bottom,
and therefore are less in contact with bacteria after 3 hours. In addition, neutrophil killing in
siliconized tubes is similar to experimental conditions used in the whole blood killing assay.
Secondly, the choice of serum or plasma seems relevant for the outcome of neutrophil killing
of GAS. However, interpretation of the data are complicated by the fact that the experiment
with serum and plasma were performed with two different neutrophils donor (Fig.11).
Therefore, an experiment should be performed that compares plasma and serum from the
same donor with the same neutrophils to pinpoint possible differences between plasma and
serum in inducing GAS killing. Nevertheless, we noticed differences in serum/plasma from
the four donors, although not correlated directly with the whole blood phenotype. As a
consequence, the importance of serum/plasma in killing differs from donor to donor and the
outcome also depends on the neutrophil donor. This could indicate that it is only possible to
reproduce the whole blood killing phenotype when plasma and neutrophils are matched, i.e.
are both obtained from the original donor; but we also must consider that whole blood is a
complex mixture of cells and soluble proteins and it might not be possible to mimic this
complex system with a simpler neutrophil/plasma assay.
On the other hand, it seems that cells are the main reason of these different whole blood
phenotypes, therefore it is possible to hypothesize that neutrophils donors are differently
important for GAS killing and that other cells might be involved in GAS killing in human
blood. Firstly, donors neutrophils might exploit different killing mechanisms to kill the
bacterium. Donors might have different expression level of antimicrobial factors, like
elastase, LL-37, azurocidin: the “Growers” and “The containing donors” might have a lower
expression of important antimicrobial molecules involved in GAS killing. This might also
support GAS in preventing the fusion of azurophilic granules with phagosomes (Staali, 2006),
and promote growth in the “Growers” hiding inside neutrophils (Medina, 2003). In addition,
this might explain the high level of whole blood phagocytosis in the “Grower” (Fig. 6C): in
this case, GAS might promote phagocytosis inside neutrophils, instead of inhibiting it (Staali,
2003), to shield from other immune defence mechanisms, and therefore survive in whole
blood. Secondly, other cells besides neutrophils might be involved like mononuclear
phagocytes (monocytes). GAS might find more difficulties to grow in “Killers” blood and it
might be easier for neutrophils to kill the bacteria. In fact, we know that GAS exploits
phagocytes cells to survive (Medina, 2003), therefore GAS might target some phagocytes,
which are not equipped to kill GAS, to promote phagocytosis inside them. As a consequence,
phagocytosis promoted by these cells would be considered not a defence mechanisms of the
host but a mechanisms exploited by GAS to shield. Therefore, the “Growers” and the
“Containing donors” monocytes might be more susceptible to GAS than the “Killers” and
support its survival inside monocytes. All these suggestions may contributes to cause the
whole blood phenotypes.

18
To conclude, blood is a very complex fluid which contains a huge variety of proteins, cells,
nutrients and metabolites and many of these factors are probably relevant for killing GAS.
Nevertheless, in our research we show that the whole blood phenotype between donors
remains regular over time, therefore all the metabolites and nutrients present in blood are not
involved in GAS survival/killing. Cells are the main reason of these different donors
phenotypes and plasma by itself is not involved, although opsonins are important for killing
GAS. Despite many mechanisms might be involved in GAS survival/killing in whole blood,
we showed important mechanisms which inhibit GAS growth and important factors which are
not involved in killing. All these findings might contribute to understand GAS pathogenesis
and the causes which lead to GAS invasive diseases in some patients.

19
Materials and methods
GAS strain
The GAS stain used for all the experiments was GAS M1T1 5448 (Kansal, 2000). Bacteria
were grown overnight in 37°C incubator. The day after bacteria were diluted 10 fold in 6 ml
THY medium and they were grown to mid-log phase till 0.4 O.D.600nm ~ 1x 108
CFU/ml.
Bacteria were collected by centrifugation, resuspended in 350 ul PBS and diluted to 0.4
O.D.600nm in 3.5 ml PBS.
Whole blood killing assay
Bacteria were diluted 10-fold in PBS. Hundred ul of 1x 107
CFU/ml bacteria and 400 ul of
human blood, collected using lepirudin as an anticoagulant, were incubated in siliconized
tubes at 37°C on a rotating device for 3 hours. Twenty ul of 10 fold serial dilutions in water
were plated on THA plates at indicated time points (0-3 hours). Plates were incubates
overnight at 37°C. Colonies were counted the day after.
Plasma survival assay
Bacteria were diluted 10-fold in PBS. Ten ul of 1x 107
CFU/ml bacteria and 40 ul of plasma
were incubated in 96-wells plate at 37°C on a shaking device (600rpm). Twenty ul of 10 fold
serial dilutions were plated on THA plates at indicated time points (0-3 hours). Plates were
incubates overnight at 37°C. Colonies were counted the day after.
FITC labeling
Bacteria were washed by centrifugation and resuspended in 10 ml PBS. Bacteria were
incubated with 0.5 mg/ml FITC solution (10 mg/ml in DMSO) on ice for 30 minutes
protected from light. Bacteria were washed 2 times with PBS and resuspended in RPMI +
0.05% HSA / PBS until required 0.4 O.D.600nm ~ 1x 108
CFU/ml.
Neutrophils numbers
Hundred and sixty ul of lepirudin anti-coagulated blood were used for experiment. Red blood
cells were lysed with 2 ml of BD FACS lysing reagent (diluted 10-fold in water), incubated
for 10 minutes at room temperature and washed away with PBS in one step of centrifugation.
Neutrophils were resuspended in 300 ul of PBS for FACS analysis. A relative number of
neutrophils was obtained collecting the events in 30 seconds within the neutrophil gate.
Whole blood phagocytosis assay
FITC labeled GAS (0.4 O.D.600nm ~ 1x 108
CFU/ml.) were diluted 10-fold in RPMI + 0.05%
HSA. Forty ul of FITC label bacteria were incubated with 160 ul of blood at 37°C for 30
minutes. Red blood cells were lysed with 2 ml of BD FACS lysing reagent (diluted 10-fold in
water), incubated for 10 minutes at room temperature and washed away with PBS in one step
of centrifugation. Neutrophils were resuspended in 300 ul of PBS for FACS analysis. A
relative number of neutrophils was obtained collecting 10,000 events within the neutrophil
gate.

20
IgG opsonization assay
Bacteria were collected by centrifugation, resuspended in 300 ul HEPES++ buffer (20 mM
HEPES, 5 mM CaCl2, 2.5 mM MgCl2, 140 mM NaCl, pH 7.3) 0.1% BSA and diluted to 0.4
O.D.600nm in 3.5 ml HEPES++ buffer 0.1% BSA. Hundred ul of the O.D. 0.4 bacterial
suspension was spun down in 1.5 ml eppendorf tube (13,000 rpm for 3 min.). The pellets were
resuspended in heat-inactivated serum, diluted in HEPES++ buffer 0.1% BSA in different
concentrations. The samples were incubated for 30 min. at 4°C, washed 1x with HEPES++
buffer 0.1% BSA and resuspended in 50 ul of protein G-alexa488 2ug/ml (obtained from
Molecular Probes). Next, the samples were incubated for 30 min on ice, washed 1x and taken
up in 300ul HEPES++ buffer 0.1% BSA. The samples were analyzed on FACS machine.
The level of fluorescence was obtained collecting 10,000 events within the gate of the
fluorescent labeled bacteria.
Neutrophil phagocytosis assay
Human neutrophils were isolated from healthy donors by using the PolymorphPrep system
(Axis-Shield). Twenty five ul of 5x107
FITC label GAS and 50 ul of donor serum or plasma
in different concentrations (diluted in RPMI + 0.05% HSA) were added to a round-bottom 96-
wells plate and incubated for 5 minutes at room temperature. Opsonized bacteria were added
to 25 ul of human neutrophils 5x106
/ml for 15 minutes at 37°C on a shaking device (600
rpm). The reaction was stopped by addition of 10 ul 10% paraformaldehyde. The samples
were analyzed on FACS.
Neutrophils killing assay
Bacteria were resuspended in RPMI instead of PBS. Human neutrophils were isolated from
healthy donors by using the PolymorphPrep system (Axis-Shield). Isolated neutrophils were
resuspended at 1x 107
neutrophils/ml in RPMI 0.05% HSA. Neutrophils were diluted to 2x
106
/ml in RPMI with 1% or 10% of HPS, active serum or plasma. For infection at an MOI of
1 (1 bacterium per neutrophil), also bacteria were diluted to 2x 106
/ml in RPMI with 1% or
10% of HPS, active serum or plasma. Hundred ul of bacterial solution and 100 ul of
neutrophils solution were incubated in siliconized tubes or round-bottom plates at 37°C on a
rotating device or shaking device for 3 hours. Twenty ul of 10 fold serial dilutions in PBS
were plated on THA plates at indicated time points (0, 30 minutes and 3 hours). Plates were
incubates overnight at 37°C. Colonies were counted the day after.

21
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