1

A capsular polysaccharide impairs microbe-guided chemotactic chasing by

neutrophils

Tamding Wangdi, Cheng-Yuk Lee, Alanna M. Spees, Chenzhou Yu, Dawn D.

Kingsbury, Sebastian E. Winter, Volkmar Heinrich and Andreas J. Bäumler

2

Text

The contribution of capsular polysaccharide to the virulence of

invasive human pathogens is commonly attributed to their anti-phagocytic

properties. Here we show that the virulence-associated (Vi) capsular

polysaccharide of Salmonella enterica serovar Typhi (S. Typhi) also

obstructs neutrophil chemotaxis, a previously overlooked property with

important consequences for host pathogen interaction. In single-cell

experiments, neutrophils extended chemotactic pseudopods toward a non-

capsulated S. Typhi mutant but not toward the capsulated S. Typhi wild

type. Formation of chemotactic pseudopods toward non-capsulated S.

Typhi required complement component 5a receptor (C5aR), suggesting that

neutrophils perceive bacteria by detecting C5a radiating from the microbial

surface. Deletion of capsule biosynthesis genes markedly enhanced uptake

of S. Typhi by neutrophils in vivo through a C5aR-dependent mechanism.

Collectively, these data suggest that the Vi capsular polysaccharide

contributes to virulence by obstructing C5a-guided chemotactic chasing of

bacteria by neutrophils.

Neutrophil chemotaxis toward intruding microbes is an essential host

defense mechanism against bacterial infection. Neutrophil migration into an

infected tissue is initially guided by chemoattractants, such as interleukin (IL)-8, a

chemokine produced by host cells after they detect the presence of bacteria

using pathogen recognition receptors (PRRs). Neutrophils that follow a gradient

3

of IL-8 to enter a site of infection will ultimately reach host cells producing this

chemokine, but this host-guided chemotaxis does not explain how neutrophils

subsequently hunt down bacteria in tissue. Early microscopic observations

suggest that chemotactic chasing of Staphylococcus aureus by neutrophils is

microbe-guided 1. A classic video produced during those studies

(http://www.biochemweb.org/neutrophil.shtml) shows that once neutrophils come

within a certain distance of S. aureus, they move directly toward bacteria by

extending chemotactic pseudopods, pushing aside red blood cells that block a

direct path. Since the identity of the chemoattractant emanating from the microbe

that guides chemotactic chasing by neutrophils remains unknown, no studies

have addressed the question whether bacteria can produce virulence factors that

can overcome this host defense mechanism.

N-formyl peptides represent one chemoattractant derived from bacteria

that might direct microbe-guided neutrophil chemotaxis 2. Alternatively, bacterial

surface carbohydrates might activate complement by the alternative pathway,

thereby giving rise to complement fragment C5a, a potent neutrophil

chemoattractant 3. The latter possibility attracted our attention, since it suggested

that inhibition of complement activation by expression of a capsular

polysaccharide could be a mechanism to evade chemotactic chasing by

neutrophils. Here we investigated this hypothesis and its consequences for host

microbe interaction in vitro and in vivo.

Salmonella enterica serotype Typhi, the causative agent of typhoid fever,

expresses a virulence-associated (Vi) capsular polysaccharide (Fig. 1A) 4. The

4

viaB locus on the S. Typhi chromosome contains genes for the regulation (tviA)

the biosynthesis (tviBCDE) and the export (vexABCDE) of the Vi capsular

polysaccharide (Fig. 1B) 5. To investigate a possible role of the Vi capsular

polysaccharide in evading neutrophil chemotaxis we compared a capsulated S.

Typhi wild-type strain (Ty2) with an isogenic non-capsulated strain lacking the

capsule biosynthesis and export genes (ΔtviB-vexE mutant) (Fig. 1A) 6. The

chemotactic response of neutrophils was first investigated using single-cell

experiments 7, in which an initially quiescent human neutrophil was picked up at

the tip of a micropipette (Fig. S1). Bacteria were immobilized by a laser optical-

trap (laser tweezers) and brought stepwise into close proximity of the neutrophil

in the presence of serum. Bringing the non-capsulated S. Typhi strain (ΔtviB-

vexE mutant) into a certain distance of a neutrophil induced a vigorous

chemotactic response, characterized by formation of a cellular pseudopod, which

protruded toward the bacteria and responded quickly to their relocation (Fig. 1C,

Supplementary video 1). In striking contrast, the capsulated S. Typhi wild-type

strain did not elicit any chemotactic response by neutrophils (Fig. 1D,

Supplementary video 2). Identical results were obtained when agglutinated

bacteria were picked up by a micropipette and brought stepwise into close

proximity of a neutrophil (Fig. 1E and F, Supplementary videos 3 and 4). These

data suggested that expression of the Vi capsular polysaccharide prevents

neutrophil chemotaxis.

Expression of the Vi capsular polysaccharide reduces complement

activation by the alternative pathway 8,9, as indicated by diminished fixation of

5

complement fragment C3b on the surface of the capsulated S. Typhi wild-type

strain compared to a non-capsulated S. Typhi strain (ΔtviB-vexE mutant) (Fig.

2A). To study the role of complement in microbe-guided chemotaxis, dual-

micropipette manipulation experiments with a non-capsulated S. Typhi strain and

neutrophils from C3-deficient mice were performed in the presence of

homologous or heterologous serum. When the assay was performed in serum

from C3-deficient mice, neutrophils did not exhibit a chemotaxic response toward

the non-capsulated S. Typhi strain (Fig. 2B, suppl. video 5). In contrast, in the

presence of serum from wild type mice (C57BL/6) neutrophils from wild type mice

(C57BL/6) and neutrophils from congenic C3-deficient mice extended cellular

pseudopods toward the bacteria (Fig. 2C and 2D; suppl. video 6 and 7). To

further investigate whether neutrophil chemotaxis elicited by a non-capsulated S.

Typhi strain was complement dependent, we monitored neutrophil migration from

the upper compartment of a Boyden chamber 10 into a bottom reservoir that

contained sterile medium or different bacterial strains. The presence of a non-

capsulated S. Typhi strain in the bottom chamber elicited migration of

significantly (P < 0.05) larger numbers of neutrophils than the presence of the

capsulated S. Typhi wild-type strain. In contrast, no neutrophil migration was

observed when the complement inhibitor Futhan 11 was added to the medium

(Fig. 2E). Collectively, these data suggested that microbe-guided neutrophil

chemotaxis is complement-dependent and can be reduced by the Vi capsular

polysaccharide.

6

Next, we investigated the mechanism by which complement directs

neutrophil chemotaxis toward bacteria. The alternative pathway of complement

activation is initiated by formation of C3 convertase, C3b fixation on the bacterial

surface and subsequent recruitment of complement components involved in

formation of the membrane attack complex, including C5. Cleavage of C5

generates the neutrophil chemoattractant C5a, which is released from the

bacterial surface 3. To investigate whether C5a emanating from the bacterial

surface was responsible for microbe-guided chemotaxis, we performed dual-

micropipette manipulation experiments with a non-capsulated S. Typhi strain and

neutrophils from C5a receptor (C5aR, also known as CD88)-deficient mice 12.

Remarkably, unlike neutrophils from wild type mice (BALB/c) (Fig. 3A, suppl.

video 8), neutrophils from C5aR-deficient mice did not exhibit any chemotactic

response toward non-capsulated bacteria (Fig. 3B, suppl. video 9). These data

suggested that neutrophils perceive bacteria by detecting a C5a gradient that

radiates from the microbial surface.

Upon cleavage from C5, C5a is quickly metabolized by carboxy-

peptidases, which remove the C-terminal arginine residue to form C5a des Arg, a

molecule with markedly reduced chemotactic activity 13. We reasoned that this

mechanism would result in the presence around a microbe of a chemotactic C5a

corona with a small diameter, which would explain why chemotactic responses

are only elicited when microbes are brought into close proximity to a neutrophil.

This hypothesis would suggest that inhibition of carboxy-peptidase activity would

increase the diameter of the C5a corona, thus increasing the maximum distance

7

between microbe and neutrophil at which a response is still induced. To test this

prediction we used zymosan-particles to model a microbial surface that activates

complement 7. By reducing the distance of a zymosan-particle to a human

neutrophil stepwise using dual-micropipette manipulation experiments, we

observed that supplementation of serum with potato carboxypeptidase inhibitor

(PCI) 14 significantly increased the maximum target perception distance (Fig. 3C

and S2) and the maximum extension of pseudopods (Fig. 3D).

Finally, we wanted to determine the biological significance of our

observations using an animal model. S. Typhi is a human restricted pathogen

and is rapidly cleared from organs of mice 8. However, since chemotactic chasing

and phagocytosis by neutrophils occurs very early during host microbe

interaction and precedes clearance of bacteria, we reasoned that these

processes could be studied at an early time point (1 hour) after injecting S. Typhi

into mice. The capsulated S. Typhi wild-type strain and a the non-capsulated S.

Typhi mutant were transformed with a plasmid encoding green fluorescence

protein (GFP) and injected intraperitoneally into mice (BALB/c). Both strains were

still present in similar numbers one hour after infection and recruited similar

numbers of neutrophils into the peritoneal cavity (Fig. S3 and S4). However, a

significantly larger fraction of neutrophils from mice infected with the non-

capsulated S. Typhi mutant were associated with bacteria compared to mice

infected with the capsulated S. Typhi wild-type strain (Fig. 3E, 3F and S3). To

determine whether these differences were due to a capsule-mediated inhibition

of C5a-guided chasing by neutrophils, congenic C5aR-deficient mice were

8

infected with the capsulated S. Typhi wild-type strain, a non-capsulated S. Typhi

mutant or vehicle control (sterile PBS). Remarkably, the fraction of neutrophils

associated with the non-capsulated S. Typhi mutant was significantly reduced in

C5aR-deficient mice and no differences were observed between capsulated and

non-capsulated bacteria. Collectively, these data suggested that C5a-guided

chemotactic chasing by neutrophils is a host defense mechanism that can be

overcome by expression of the Vi capsular polysaccharide.

Chemotactic chasing by neutrophils was first observed over 50 years ago

1, but the phenomenon remains understudied, in part because most assays

investigating the interaction of microbes with phagocytes do not disentangle this

property from subsequent phagocytic events 15. Furthermore, conventional cell

migration assays, such as the Boyden chamber assay, lack the resolution to

distinguish between host-guided chemotaxis and microbe-guided chemotaxis.

The recent development of single-cell experiments provides now a convenient

assay for studying microbe-guided chemotaxis 15. Using this approach we show

that expression of the Vi capsular polysaccharide inhibits C5a-guided

chemotaxis, suggesting that this virulence factor restrains chemotactic chasing

by neutrophils. Our supplementary videos illustrate that through this mechanism

the Vi capsular polysaccharide can act as a “cloaking device” that makes S.

Typhi practically “invisible” to neutrophils, thereby enabling the pathogen to

evade this arm of the host defense in vivo (Fig. 3E and 3F). This previously

unrecognized property of the Vi capsular polysaccharide is likely relevant to the

9

interaction of our immune system with many other pathogenic microbes that

evade complement activation.

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14. Ryan, C.A., Hass, G.M. & Kuhn, R.W. Purification and properties of a

carboxypeptidase inhibitor from potatoes. J Biol Chem 249, 5495-5499

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15. Heinrich, V. & Lee, C.Y. Blurred line between chemotactic chase and

phagocytic consumption: an immunophysical single-cell perspective. J

Cell Sci 124, 3041-3051 (2011).

11

16. Cummings, L.A., Wilkerson, W.D., Bergsbaken, T. & Cookson, B.T. In

vivo, fliC expression by Salmonella enterica serovar Typhimurium is

heterogeneous, regulated by ClpX, and anatomically restricted. Mol

Microbiol 61, 795-809 (2006).

17. Raffatellu, M., et al. The capsule encoding the viaB locus reduces

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12

Supplementary Information accompanies the paper on www.nature.com/nature.

Acknowledgements

This project was supported by Public Health Service grant AI044170 to AJB. We

would also like to acknowledge the NIH Facilities Infrastructure Grant 1-C06-

RR12088-01. The authors do not declare conflicts of interest.

Author Information

Affiliations

Department of Medical Microbiology and Immunology, School of Medicine,

University of California at Davis, One Shields Ave; Davis CA 95616, USA

Tamding Wangdi, Alanna M. Spees, Dawn D. Kingsbury, Sebastian E. Winter,

and Andreas J. Bäumler

Department of Biomedical Engineering, University of California at Davis, One

Shields Ave; Davis CA 95616, USA

Cheng-Yuk Lee, Chenzhou Yu, Volkmar Heinrich

Contributions

13

TW contributed to the experimental design and contributed to Figures 1A, 1C,

1D, 1E, 1F, 2A, 2B, 2C, 2D, 2E, 3A, 3B, 3C, 3D, 3E, 3F, S3, S4A, S4B and S4C.

A.M.S. contributed to Figures 3E, 3F, S3, S4A, S4B and S4C. D.D.K. contributed

to Figures to Figures 3E, 3F, S3, S4A, S4B and S4C. S.E.W. constructed

bacterial plasmids and critically read the manuscript. C.-Y.L. contributed to

Figures 1E, 1F, 3B, 3C, 3D, 3A and 3B. C.Y. contributed to Figures 1C and 1D.

V.H. contributed to Figures S1 and S2. A.J.B. contributed to Figure 1B. A.J.B.

and V.H. provided financial support for the study and contributed to the

experimental design. A.J.B. and V.H. contributed equally to this work.

Competing financial Interests

The authors declare no competing financial interests

Correspondence

Correspondence to: Andreas J. Bäumler (ajbaumler@ucdavis.edu).

14

METHODS SUMMARY

The S. Typhi wild-type isolate Ty2 was obtained from the American Type

Culture Collection. A derivative of Ty2 carrying a ΔtviB-vexE deletion has been

described previously 6. To label bacteria with GFP, S. Typhi strains were

transformed with plasmid pDW5 16. All animal experiments were performed

according to Association for Assessment and Accreditation of Laboratory Animal

Care (AAALAC) guidelines and approved by the Institutional Animal Care and

Use Committee at the University of California at (UC) Davis. The UC Davis

Institutional Review Board approved the protocol for obtaining blood draws and

informed consent was obtained from all individuals. Six to eight week old female

mice obtained from The Jackson Laboratory were used for this study. Mice were

injected intraperitoneally with 1 x 107 colony forming units (CFU)/animal

suspended in 0.1 ml phosphate buffered saline (PBS). A neutrophil enrichment

kit (Stemcell Technologies, Vancouver, Canada) was used to collect murine

neutrophils. Human neutrophils were isolated using the Cytoselect Cell Migration

Assay kit (Cellbiolabs Inc., San Diego, CA). Neutrophils were suspended in

chemotaxis medium (1.5x106 cells/ml in Hank's Balanced Salt Solution with 10%

autologous serum). Rabbit anti-Vi serum (for Ty2) or rabbit anti-O9 serum (for

ΔtviB-vexE mutant) were used to generate bacterial micro-agglutinates. To

measure the maximum target perception distance, zymosan (St. Louis, MO)-

particles were prepared as described previously 7. To inhibit cleavage of C5a, 36

μg/ml of potato carboxypeptidase inhibitor (PCI) 14 (Sigma, St. Louis, MO) were

added to the chemotaxis medium. The micropipette setup has been described

15

previously 7. Vi capsule expression and C3b deposition were detected using flow

cytometry as described previously 8,17. Other flow cytometry experiments and the

Boyden chamber assay are described in the supplementary methods. To

determine statistical significance an unpaired Student t-test was used.

16

Figure Legends

Figure 1: The Vi capsular polysaccharide inhibits microbe-guided neutrophil

chemotaxis.

(A) Expression of the Vi capsular polysaccharide (Y-axis) was detected in

cultures of the S. Typhi wild type (wt, left panel) and a non-capsulated S. Typhi

strain (ΔtviB-vexE mutant, right panel) by flow cytometry using rabbit anti-Vi

serum. (B) Schematic drawing of Vi capsular biosynthesis genes (black arrows)

of S. Typhi. (C-F) The indicated bacterial strains were immobilized by laser

tweezers (arrows in C and D) or agglutinated with rabbit serum and picked up

with a micropipette (E and F). Bacteria were brought in close proximity to a

pipette-held human neutrophil and videomicrographs were taken at the indicated

time points. At least four neutrophils were analyzed per blood sample and each

experiment was repeated with neutrophils from different individuals (N = 4). (C

and E) Note chemotactic pseudopods that extend from neutrophils toward

bacteria.

Figure 2: The Vi capsular polysaccharide inhibits complement-dependent

neutrophil chemotaxis.

(A) Fixation of C3b (Y-axis) on the surface of the S. Typhi wild type (wt, left

panel) or a non-capsulated S. Typhi strain (ΔtviB-vexE mutant, right panel) that

had been incubated for 30 minutes in 1% human serum was detected by flow

cytometry using FITC-conjugated goat anti-human C3b antibody. (B-D)

17

Videomicrographs of dual-micropipette manipulation experiments with an

agglutinated non-capsulated S. Typhi strain and murine neutrophils from wild

type (C57BL/6) mice (D) or congenic C3-deficient mice (B and C) performed in

buffer containing serum from wild type mice (C and D) or in serum from C3-

deficient mice (B). For each strain, blood from 4 animals was pooled for isolation

of serum and neutrophils for an experiment. Each experiment was repeated at

least four times. (E) Neutrophil migration into the bottom compartment of a

Boyden chamber containing vehicle control (HBSS) or the indicated bacterial

strains. Bars represent averages ± standard error from 4 donors. **, P < 0.01.

Figure 3: The Vi capsular polysaccharide inhibits C5aR-dependent neutrophil

chemotaxis.

(A and B) Videomicrographs of dual micropipette manipulation experiments with

an agglutinated non-capsulated S. Typhi strain (ΔtviB-vexE mutant) and murine

neutrophils from wild type (BALB/c) mice (A) or congenic C5aR-deficient mice

(B). For each strain, blood from 4 animals was pooled for isolation of serum and

neutrophils for an experiment. Each experiment was repeated at least four times.

(C and D) Maximum target perception distance (C) and the maximum extension

of pseudopods (D) was determined using dual-micropipette manipulation of a

zymosan-particle and a human neutrophil in the presence or absence of potato

carboxypeptidase inhibitor (PCI). Figure represents experiments with blood from

two donors, with at least four neutrophils being tested each per experimental

condition. (E and F) Mice were infected intraperitoneally with the indicated GFP-

18

labeled bacterial strains and cells were collected one hour later by intraperitoneal

lavage. Representative images of bacterial association (Y-axis) with neutrophils

from wild type mice or congenic C5aR-deficient mice (E) and quantitative

analysis of the data (averages ± standard error) from groups of six animals (F)

are shown. (C, D and F) **, P < 0.01; *, P < 0.05; ns, not significantly different.

Figure S1: Single-cell approach to assess neutrophil chemotaxis toward S. Typhi

in buffer containing serum.

S. Typhi (image on the top left) and neutrophils from humans or mice (images on

the top right) were deposited into a microscopy chamber with two open sides

(schematic drawing in the center). Vertically adjustable water reservoirs allowed

accurate pressure application to facing micropipettes inserted into this chamber.

An initially quiescent neutrophil was aspirated at the tip of a micropipette. S.

Typhi was either immobilized with optical tweezers or micro-aggregates of

bacteria were produced and picked up using a second micropipette. Bacteria and

neutrophils were then brought stepwise into close proximity (image at the

bottom).

Figure S2: Measurement of the maximum target perception distance and the

maximum extension of pseudopods.

Using dual-micropipette manipulation, a zymosan-particle was stepwise brought

into close proximity of an initially passive human neutrophil. Relative times are

indicated in the videomicrographs. Images illustrate the determination of the

19

average target distance at which pseudopod formation started within 2 minutes

(first videomicrograph from the left) and the maximum length of chemotactic

pseudopods (fourth videomicrograph from the left).

Figure S3: Gating strategy for detecting an association of S. Typhi with

neutrophils from peritoneal lavage.

Gating strategy for analyzing peritoneal lavage cell suspensions. After doublet

elimination (top left panel) live cells were gated (top middle panel) and CD3+

B220+ NK1.1+ cells eliminated using a dump channel (top right panel). CD3-

B220- NK1.1- cells were then analyzed for expression of CD11B (right panel in

the middle row). Next, CD3- B220- NK1.1- CD11B+ cells were analyzed for

expression of LY6C and LY6G cells (left panel in the middle row). CD3- B220-

NK1.1- CD11B+ LY6C+ LY6G+ cells (neutrophils) were analyzed by microscopy to

confirm neutrophil morphology (bottom right panel). The gate for the detection of

GFP-labeled S. Typhi in neutrophil populations was set using cells isolated from

control mice injected with sterile PBS (bottom left and middle panels).

Figure S4: Quantification of neutrophil infiltrates elicited by infection with S.

Typhi.

Mice (BALB/c) were injected intraperitoneally with sterile PBS or the indicated

GFP-labeled bacterial strains and cells were collected one hour later by

intraperitoneal lavage. (A) Representative images of neutrophil infiltration (LY6C+

LY6G+ cells) in intraperitoneal lavage populations detected by flow cytometry. (B)

20

Quantitative analysis of neutrophil infiltration is shown as geometric means (bars)

± standard error from groups of six animals. (C) Bacterial numbers recovered

from peritoneal lavage. NS, not significantly different.

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