ArticlePDF Available

Infection by Tubercular Mycobacteria Is Spread by Nonlytic Ejection from Their Amoeba Hosts

Authors:
Monica Hagedorn at Constructor University Bremen gGmbH
Monica Hagedorn
Kyle H Rohde at University of Central Florida
Kyle H Rohde
David G Russell at Cornell University
David G Russell
Thierry Soldati at University of Geneva
Thierry Soldati
Download full-text PDF
Read full-text
Download citation

Abstract and Figures

To generate efficient vaccines and cures for Mycobacterium tuberculosis, we need a far better understanding of its modes of infection, persistence, and spreading. Host cell entry and the establishment of a replication niche are well understood, but little is known about how tubercular mycobacteria exit host cells and disseminate the infection. Using the social amoeba Dictyostelium as a genetically tractable host for pathogenic mycobacteria, we discovered that M. tuberculosis and M. marinum, but not M. avium, are ejected from the cell through an actin-based structure, the ejectosome. This conserved nonlytic spreading mechanism requires a cytoskeleton regulator from the host and an intact mycobacterial ESX-1 secretion system. This insight offers new directions for research into the spreading of tubercular mycobacteria infections in mammalian cells.
Figures - uploaded by Thierry Soldati
Author content
All figure content in this area was uploaded by Thierry Soldati
Content may be subject to copyright.
Content uploaded by Thierry Soldati
Author content
All content in this area was uploaded by Thierry Soldati
Content may be subject to copyright.
Infection by tubercular mycobacteria is spread by nonlytic
ejection from their amoeba hosts
Monica Hagedorn1, Kyle H. Rohde2, David G. Russell2, and Thierry Soldati1,*
1Département de Biochimie, Faculté des Sciences, Université de Genève, Sciences II, 30 quai
Ernest Ansermet, CH-1211-Genève-4, Switzerland. 2Microbiology and Immunology, College of
Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
Abstract
To generate efficient vaccines and cures for Mycobacterium tuberculosis, we need a far better
understanding of modes of infection, persistence and spreading. Host cell entry and establishment
of a replication niche are well understood, but little is known about how tubercular mycobacteria
exit host cells and disseminate the infection. Using the social amoeba Dictyostelium as a genetically
tractable host for pathogenic mycobacteria, we discovered that M. tuberculosis and M. marinum but
not M. avium are ejected from the cell through an actin-based structure, the ejectosome. This
conserved nonlytic spreading mechanism requires a cytoskeleton regulator from the host and an intact
mycobacterial ESX-1 secretion system. This insight offers new directions for research into the
spreading of tubercular mycobacteria infections in mammalian cells.
Keywords
Mycobacterium marinum; Infection; nonlytic release
Intracellular bacterial pathogens have evolved strategies to exploit host cell resources and
replicate inside a variety of cell types, staying out of reach of the host’s immune system. The
concept is emerging that pathogenic bacteria evolved from environmental species by adapting
to an intracellular lifestyle within free-living, bacteria-eating protozoans. Consequently,
cellular defense mechanisms active in animal immune phagocytes may have originated in
amoebae (1,2). For example, during differentiation of the social amoeba Dictyostelium to form
a multicellular structure, Sentinel cells are deployed to combat pathogens (1).
Generally, infection follows entry of a bacterium inside a host cell, giving rise to a pathogen-
containing vacuole usually called a phagosome. From this common starting point, pathogens
subvert or resist the mechanisms that usually transform the phagosome into a bactericidal
environment. Understanding of the passive or triggered uptake mechanisms and of the
subsequent hijacking of host cell processes is increasing steadily, but little is known about how
the pathogens exit their primary host cell and spread the infection.
Mycobacterium tuberculosis causes tuberculosis and other granulomatous lesions and is a
major threat to human health. M. marinum is a close relative (3) responsible for fish and
*To whom correspondence should be addressed. thierry.soldati@unige.ch.
One-sentence summary: Mycobacteria exit host cells without lysing them via an ejectosome generated by the concerted action of host
and pathogen factors.
NIH Public Access
Author Manuscript
Science. Author manuscript; available in PMC 2009 October 29.
Published in final edited form as:
Science. 2009 March 27; 323(5922): 1729–1733. doi:10.1126/science.1169381.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
amphibian tuberculosis, in which it causes almost indistinguishable pathologies and lesions
[reviewed in (4)]. Several elegant cross-species complementation studies between these two
pathogens highlight their common mechanisms of pathogenicity [reviewed in (4)].
After passive uptake by immune phagocytes, M. tuberculosis and M. marinum arrest or bypass
phagolysosome maturation and replicate inside a compartment of endosomal nature (57). Both
species can escape from this vacuole into the host cytosol (8–11), though at varying frequencies.
Efficient translocation into the cytosol depends on an intact region of difference (RD) 1 locus
(10,11), which encodes components of a type seven secretion system and essential secreted
effectors (12). This ESX-1 secretion system has been implicated in arrest of phagosome
maturation (13), granuloma formation and the spread of infection (14,15); however, it is not
essential for replication inside macrophages (14). It has also been directly associated with the
secretion of a membranolytic activity (15). Specifically, the secreted effector ESAT-6 has
recently been linked to niche breakage and pore-forming activity in macrophages (11).
M. marinum and M. tuberculosis can disseminate through release of bacilli following host cell
lysis via necrotic or apoptotic cell death (16–18), but studies also document cell-to-cell,
antibiotic-insensitive spreading inside an epithelial monolayer (19,20). M. marinum induces
plasma membrane protrusions (2,9) suggested to participate in dissemination between
macrophages in culture (21) and inside zebrafish embryos (22). Hence, escape into the cytosol
may be a necessary precursor for cell-to-cell spread and may involve a direct nonlytic
transmission process occuring within the granuloma (5,6).
Here, we adopted the genetically tractable Dictyostelium-M. marinum model system to unravel
basic mechanisms of intercellular dissemination. The course of M. marinum infection in
Dictyostelium amoebae is very similar to that in macrophages (8). The mycobacteria replication
vacuole accumulates a flotillin-like raft protein, then ruptures and releases M. marinum into
the cytosol (see fig. S1). A Dictyostelium mutant lacking the RacH GTPase, which is involved
in regulation of the actin cytoskeleton and endosomal membrane trafficking and acidification,
was more permissive for M. marinum proliferation (8). Detailed FACS analysis (fig. S2) of
these infected cells suggested an interference with intercellular dissemination (fig. S3).
To test this hypothesis, we designed a quantitative dissemination assay (Fig. 1A). Briefly, an
infected Dictyostelium donor strain (either wild type or racH-) is mixed with a green fluorescent
wild type acceptor strain at a donor:acceptor ratio of 1:5. Over the course of infection, the
number of bacteria per donor and acceptor cell was determined by visual inspection. The
proportion of wild type infected donor cells decreased concomitantly with a sharp increase of
infected acceptor cells at 21 hours post infection (hpi), and the number of bacteria per acceptor
cell increased over time (Fig. 1B, bar shadings). This indicated successful transmission of
bacteria and replication in acceptor cells. In contrast, the proportion of infected racH-donor
cells remained relatively constant (above 50%). Strikingly, infection of acceptor cells from
racH-donor cells was about 8-fold less than from wild type donor cells (Fig. 1C). The racH-
cells were deficient in intercellular spreading of mycobacteria and it appears that a
RacHdependent release mechanism is required for cell-to-cell transmission under these
conditions.
Cytosolic pathogens, such as Listeria and Shigella use actin-based tails and filopodia for
intercellular spreading. During M. marinum infection of macrophages, unidentified
mycobacterial proteins induce actin tails in a Wiscott Aldrich Syndrome protein (WASP)
dependent manner (23), as well as structures reminiscent of Shigella-induced filopodia (9) that
can be captured by neighboring cells ((9) and fig S4). Hence, we monitored F-actin dynamics
in infected cells expressing a GFP-fusion with the actin binding domain (ABD) of filamin
(24) by live microscopy (Movies S1–S7,Fig. 1D–F and fig. S5A,B). At late stages of infection,
Hagedorn et al. Page 2
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
despite the presence of many cytosolic bacteria, cells exhibited apparently normal amoeboid
motility (Movie S1) and cell division (Movie S2). In contrast to the observation in macrophages
((9) fig. S4), no persistent actin tails were visible on cytosolic bacteria, possibly due to the high
rate of actin depolymerisation in Dictyostelium (25). However, transient actin flashes were
produced when bacteria contacted the cell cortex (fig. S5A, Movie S3).
Bacteria were “ejected” through an F-actin-dense structure we called an “ejectosome” (Fig.
1D, Movie S4). Mycobacteria were also caught “spanning” the plasma membrane without
inducing host cell lysis, a situation stable for the duration of the observation (e.g. 17 min, Fig.
1E, Movie S5). A significant proportion of cells harbored multiple similar structures (fig. S5B,
Movie S6). Ejectosomes can apparently exert a contractile force, forming a tight septum around
the bacteria as they are towed behind motile host cells (Fig. 1D, Movie S4). We also captured
images of synchronized ejection from a donor cell and phagocytosis by an acceptor cell (Fig.
1F, Movie S7). Hence, an actin-based mechanism appeared to be responsible for nonlytic
ejection of cytosolic mycobacteria, which, concomitantly with capture by neighboring cells,
ensured efficient and antibiotic-insensitive intercellular spreading.
The live observations were confirmed by staining fixed infected cells with fluorescent
phalloidin. During phagocytosis, the cell deforms towards the bacteria and extends
lamellipodia that surround it along most of its length (Fig. 2A, 3E). By contrast, ejectosomes
are short barrel-shaped structures usually found in flat membrane regions (Fig. 2B). Multiple
ejectosomes were often observed to cluster (Fig. 2C, D). Cell fixation preserved the structure
of ejectosomes “coupled” to phagocytic cups suggesting direct donor-to-acceptor transmission
(Fig. 2E).
What propels bacteria during ejection is unclear, because we rarely observed bacteria with
typical F-actin-tails (Fig. 1G). Ejection may be powered by a mechanical process resulting
from a combination of cortical tension, cytoplasmic pressure and a reaction of the actin-cortex
to bacteria-induced deformation. Myosin IB and coronin were strongly enriched at the
ejectosome (fig. S5G, H), whereas myosin II and the Arp2/3 complex were not or weakly
enriched in the Factin barrel (fig. S5I, J). Thus, the ejectosome may either assemble de novo
or result from a rearrangement of preexisting cortical structures.
In all cases of ejection, the part of the bacterium inside the cell was devoid of membrane markers
from endosomes (Fig. 2F and fig. S5C) or the replication compartment (Fig. 2F, G), which
excludes the phenomenon being an exocytic event. The protruding part of the bacterium
induced a bulge in the plasma membrane (Fig. 2F, fig. S5C), demonstrating that the bacterium
is on an outward journey through the cell surface. The membrane labeling often appeared
patchy at the tip of the bulge (Fig. 2F, fig. S5C), suggesting local loss of integrity. Sometimes,
extracellular mycobacteria were wrapped in plasma membrane remnants (Fig. 2H, fig. S5D).
Outward plasma membrane deformation (Fig. 3A, C) and partial membrane rupture (Fig. 3B,
C) were also observed by scanning and transmission electron microscopy.
Labelling of the protruding part of ejected M. marinum with an anti-M. marinum serum in non-
permeabilised cells indicated accessibility of the bacterium surface, and hence local loss of
plasma membrane integrity (Fig. 2I, K). However, infected cells do not lyse, as judged by live
imaging (Fig. 1E, Movies S1–S7), and they are also not leaky, as demonstrated by the exclusion
of a membrane-impermeant DNA-binding dye during a two hours-long incubation (fig. S5E).
Serial sections through an ejectosome (Fig. 3C) showed a cytosolic bacterium protruding from
a cell with the plasma membrane ruptured towards the tip of the bacterium, but at the same
time invaginated between the bacterium and a zone of dense actin meshwork. We propose that,
during ejection, the ingressing plasma membrane stays tightly apposed to the bacterium surface
Hagedorn et al. Page 3
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
and finally reseals at the posterior of the bacterium, resulting in a dynamic but tight seal that
prevents host cell lysis or leakage (schematically depicted in Fig. 3D).
During Dictyostelium infections, M. tuberculosis was first found in spacious vacuoles (fig. S7)
that accumulated vacuolin, the Dictyostelium flotillin homologue (fig. S6A). Then, with an
efficiency lower than that of M. marinum, the bacilli translocated into the cytosol (Fig. 4A, fig.
S6A, S7) where they induced ejectosomes (Fig. 4A, fig. S6B, C). In Dictyostelium, M.
avium also accumulated in spacious compartments decorated with vacuolin, but, in contrast to
M. tuberculosis and M. marinum, no vacuole breakage was detected (Fig. 4A), and no
ejectosome was observed. Vacuole escape and nonlytic ejection from the host cell may
represent conserved strategies among tubercular mycobacteria that could play a prominent role
in dissemination of infection, rather than intracellular survival per se.
We expected a direct correlation between the observed number of ejectosomes and the
spreading of infection. Indeed, in wild type cells, there were 14–15 ejectosomes/100 cells at
37 hpi, whereas no ejectosome was ever observed in racH-cells (Fig. 4B, fig. S8B). This is not
due to deficient vacuole escape, because at 37 hpi, over 90 % of M. marinum were cytosolic
and free of vacuolin staining, compared to 75% in wild type cells (Fig. 4B, fig. S9).
Complementation of racH-cells with GFP-RacH partially restored the capacity to form
ejectosomes (fig. S8C). In accordance, membrane fragments carrying GFP-RacH polymerize
actin around them upon addition of GTPγS in a cell-free assay (26).
In correlation with the capacity to induce ejectosomes, the RD1 locus is present in M.
tuberculosis and M. marinum (13–15), but absent from M. avium (12). A M. marinum mutant
deleted of its RD1 locus (ΔRD1) replicated inefficiently in Dictyostelium (Fig. 4D), and the
population of infected cells steadily decreased with time (Fig 4E and fig. S8D). Vacuole escape
was readily detectable (Fig. 4B and fig. S9), but was about 4-to 5-fold less efficient than for
wild type M. marinum, as measured by colocalization with vacuolin or p80. This corroborates
recent findings in macrophages using a collection of ESX-1 mutants (11). But the most striking
observation was the complete absence of ejectosomes (Fig. 4B). If the ESX-1 locus secretes
vacuole escape factors (11) it may also secrete factors that coordinate egress of the resulting
cytosolic bacteria. It is possible that ESAT-6, one of the major secreted effectors, plays a role
in both processes. We designed a trans-complementation strategy in which M. marinum
ESAT-6 was conditionally expressed directly inside the cytosol of its host Dictyostelium (Fig.
4C). M. marinum ΔRD1 apparently replicated better in ESAT-6 expressing cells than in wild
type cells (Fig. 4D) and showed a 1.5-to 2-fold increased frequency of niche escape. Most
importantly, it induced ejectosome formation (Fig. 4B). Our data demonstrated that nonlytic
ejection of tubercular mycobacteria from Dictyostelium is a concerted process that necessitates
host and pathogen factor(s), and is crucial for the maintenance of an infection in a cell
population.
Conceptually, the ejectosome bears intriguing analogies with the contracting actin-ring formed
during purse-string closure of membrane wounds in Xenopus oocytes (27) and during the repair
of toxin-induced macroapertures in endothelial cells (28). Because lytic release from a single-
celled amoeba would be lethal, the ejectosome may have first evolved as a plasma membrane
repair mechanism. This concerted and mutually benefitial strategy might have been conserved
during evolution to ensure dissemination of mycobacteria between immune phagocytes of their
metazoan host.
Dictyostelium can be thought of as a rudimentary innate immune phagocyte and this model has
allowed us to identify a conserved strategy for egress and cell-to-cell spread that is shared by
M. marinum and M. tuberculosis.
Hagedorn et al. Page 4
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
References
1. Chen G, Zhuchenko O, Kuspa A. Science (New York, N.Y 2007 Aug 3;317:678.
2. Stamm LM, Brown EJ. Microbes Infect 2004 Dec;6:1418. [PubMed: 15596129]
3. Stinear TP, et al. Genome Res 2008 May;18:729. [PubMed: 18403782]
4. Tobin DM, Ramakrishnan L. Cell Microbiol 2008 May;10:1027. [PubMed: 18298637]
5. Russell DG. Nat Rev Microbiol 2007 Jan;5:39. [PubMed: 17160001]
6. Cosma CL, Sherman DR, Ramakrishnan L. Annu Rev Microbiol 2003;57:641. [PubMed: 14527294]
7. Rohde KH, Abramovitch RB, Russell DG. Cell Host Microbe 2007 Nov 15;2:352. [PubMed:
18005756]
8. Hagedorn M, Soldati T. Cell Microbiol 2007 Nov;9:2716. [PubMed: 17587329]
9. Stamm LM, et al. J Exp Med 2003 Nov 3;198:1361. [PubMed: 14597736]
10. van der Wel N, et al. Cell 2007 Jun 29;129:1287. [PubMed: 17604718]
11. Smith J, et al. Infect Immun. 2008 Oct 13;
12. Abdallah AM, et al. Nat Rev Microbiol 2007 Nov;5:883. [PubMed: 17922044]
13. Tan T, Lee WL, Alexander DC, Grinstein S, Liu J. Cell Microbiol 2006 Sep;8:1417. [PubMed:
16922861]
14. Volkman HE, et al. PLoS biology 2004 Nov;2:e367. [PubMed: 15510227]
15. Gao LY, et al. Mol Microbiol 2004 Sep;53:1677. [PubMed: 15341647]
16. Derrick SC, Morris SL. Cell Microbiol 2007 Jun;9:1547. [PubMed: 17298391]
17. Chen M, Gan H, Remold HG. J Immunol 2006 Mar 15;176:3707. [PubMed: 16517739]
18. Davis JM, Ramakrishnan L. Cell 2009 Jan 9;136:37. [PubMed: 19135887]
19. Byrd TF, Green GM, Fowlston SE, Lyons CR. Infect Immun 1998 Nov;66:5132. [PubMed: 9784514]
20. Castro-Garza J, King CH, Swords WE, Quinn FD. FEMS Microbiol Lett 2002 Jul 2;212:145.
[PubMed: 12113926]
21. Carlsson F, Brown EJ. J Cell Physiol 2006 Nov;209:288. [PubMed: 16826602]
22. Davis JM, et al. Immunity 2002 Dec;17:693. [PubMed: 12479816]
23. Stamm LM, et al. Proc Natl Acad Sci U S A 2005 Oct 11;102:14837. [PubMed: 16199520]
24. Lee E, Knecht DA. Traffic 2002 Mar;3:186. [PubMed: 11886589]
25. Zigmond SH. Cell Motil Cytoskeleton 1993;25:309. [PubMed: 8402952]
26. Somesh BP, Neffgen C, Iijima M, Devreotes P, Rivero F. Traffic 2006 Jun 29;7:1194. [PubMed:
17004322]
27. Mandato CA, Bement WM. J Cell Biol 2001 Aug 20;154:785. [PubMed: 11502762]
28. Boyer L, et al. J Cell Biol 2006 Jun 5;173:809. [PubMed: 16754962]
29. Acknowledgements. We gratefully acknowledge Lalita Ramakrishnan and Christine Cosma for
providing strains of M. marinum, various GFP-expression vectors and advice; Gareth Griffiths for
GFP-expressing M. avium; Brian C. VanderVen for providing GFP-expressing M. tuberculosis;
Francisco Rivero and Markus Maniak for providing Dictyostelium mutant strains; Christoph Bauer
of the NCCR imaging platform for his help with microscopy; Paul Walther and Eberhard Schmid for
their expert help with the SEM; Dominique Soldati for critical reading of the manuscript. The work
was supported by the Swiss National Science Foundation in the form of a grant to TS and an individual
short-term fellowship to MH. The TS group participates in the NEMO (non-mammalian experimental
models for the study of bacterial infections) network supported by the Swiss 3R Foundation. DGR
and KHR are supported by grants AI 067027 and HL 055936 from the US National Institutes of
Health.
Hagedorn et al. Page 5
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 1.
Direct cell-to-cell transmission of M. marinum occurs via ejectosomes and is RacH dependent.
(A) Quantitative dissemination assay. A donor strain (wild type or racH-cells) was infected
with DsRed-expressing M. marinum and, at 12 hpi, mixed with a green fluorescent acceptor
strain. Presence of bacteria in donor and acceptor cells was scored. Dissemination efficiency
for wild type (B) and racH-(C) donor cells. Number of bacteria per cell (classified into groups
of 1–3, 4–10 and >10 bacteria/cell) are indicated. (D–F) Live GFP-ABD expressing
Dictyostelium cells (green) infected with DsRed-expressing M. marinum (red) were imaged at
35 hpi for the indicated times (upper left corner). (D) Nonlytic ejection of mycobacteria occurs
through actin-dense ejectosomes (white arrowhead). (E) Cells with mycobacteria spanning the
Hagedorn et al. Page 6
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
plasma membrane retain normal motility. (F) Cell-to-cell transmission of a cytosolic bacterium
(black arrowhead) from a donor cell (do) to an acceptor cell (ac) through an ejectosome (white
arrowhead) into a phagocytic cup (white arrows). (G) A bundle of bacteria are ejected from a
donor cell (do). The plasma membrane bulge (small arrows) is ruptured at the tip (asterisk),
where it contacts an acceptor cell (ac). Actin tails stained by phalloidin (green, arrowheads),
were polarized at the posterior of bacteria (blue). Vertical distance from the first section is
indicated in µm. Scale bars 1 µm.
Hagedorn et al. Page 7
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 2.
Biogenesis, structure and topology of ejectosomes. Paraformaldehyde-fixed cells infected with
GFP-expressing M. marinum (green), stained for F-actin (red). (A) A phagocytic cup lined
with F-actin (arrows). A single (B) or multiple bacteria (C, D) spanning the host cell plasma
membrane (asterisks) through ejectosomes (white arrowheads) with the intracellular part of
the bacterium devoid of actin-labeling (black arrowhead). (E) Cell-to-cell transmission of
bacteria from a donor (do) to an acceptor (ac) cell through an ejectosome into a phagocytic cup
(arrows). (F) Movement of cytosolic bacteria (blue) through ejectosomes (F-actin, green)
induces a plasma membrane bulge (small arrows) that ruptures at the tip (F, asterisk). The
intracellular part of the ejecting bacterium is devoid of p80, a plasma membrane marker (F),
Hagedorn et al. Page 8
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
and vacuolin, a niche marker (asterisk, G). Some extracellular bacteria were positive for p80
(small arrows, H). (I–K) Live infected cells (M. marinum, blue, actin, green) were incubated
in the presence of an anti-M. marinum serum. The extracellular parts of ejecting (small arrows,
I, K) or outside (small arrow, J) bacteria were accessible to the antibody and labeled red, in
contrast to intracellular bacteria (black arrowhead, J), confirming partial loss of plasma
membrane integrity. Scale bars 1 µm.
Hagedorn et al. Page 9
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 3.
Electron microscopy of ejecting bacteria and schematic representation of an ejection event.
Scanning electron microscopy (A–B, A’ is the magnified inset of A) showed bulges of the
plasma membrane (small arrows), some ruptured at the tip of the ejecting bacterium (B,
asterisk). Scale bars 0.5 µm. (C) Serial sections through an ejectosome revealed the
organization of the Factin (white arrowheads) and the plasma membrane. The posterior of the
ejecting bacterium (black arrowhead) was in the cytosol. The bacterium was separated from
the F-actin by the invaginated plasma membrane, which was tightly apposed to its surface.
Membrane fragments were scattered along the extracellular part of the bacterium (small
arrows). (D) Schematic representation of an ejection. During outward movement of a bacterium
(green), the F-actin barrel exerts contraction (red arrows), and the invaginating plasma
Hagedorn et al. Page 10
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
membrane (PM) reseals at the posterior of the bacterium (black arrows), maintaining a tight
septum despite partial membrane rupture. (E) Micrograph of a phagocytic cup. The actin-filled
lamellipodia (white arrowheads) extend and engulf a bacterium. Scale bars 1µm.
Hagedorn et al. Page 11
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Fig. 4.
Ejection is a process involving both host and pathogen factors and is a strategy shared by
tubercular mycobacteria. (A) In wild type cells (Dd wt) both, M. marinum (mar) and M.
tuberculosis (tuber) were able to escape their vacuolin–coated (green) niche (upper row, black
arrowheads) and form ejectosomes (lower row, green, white arrowheads). Plasma membrane
fragments on ejecting bacteria were positive for p80 (red, small arrows), but the intracellular
part of the bacteria was devoid of p80 labeling (black arrowhead). In contrast M. avium
remained in a vacuolin-positive compartment (upper row) and did not form ejectosomes. (B)
In racH-cells GFP-expressing M. marinum (red) translocated into the cytosol (upper row), but
no ejectosome was detected (lower row; actin, green; p80, red). M. marinum ΔRD1 escaped
Hagedorn et al. Page 12
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
its niche in wild type and ESAT-6 (E6) expressing cells; however, ejectosomes (white
arrowhead) were only detected in ESAT-6 expressing cells. (C–E) Trans-complementation by
expression of M. marinum ESAT-6 in the Dictyostelium cytosol. Removal of tetracycline-
induced ESAT-6 expression (C). Wild type and ESAT-6 expressing cells were infected with
GFP-expressing M. marinum ΔRD1 and the infection monitored by FACS. Quantification of
total fluorescence of infected cells indicated increased replication of M. marinum ΔRD1 in
ESAT-6 expressers (D, red curve, normalized to T=0.5 hpi). Infected ESAT-6 expressers with
high fluorescence (FL1) persisted longer (E, red curve), compared to infected wild type
Dictyostelium (D, E, blue curve).
Hagedorn et al. Page 13
Science. Author manuscript; available in PMC 2009 October 29.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
... The genetically tractable amoeba Dictyostelium discoideum, a professional phagocyte, is well established as a useful model system to dissect the basic interactions between host cells and their pathogens, such as M. marinum (Barisch & Soldati, 2017;Hagedorn & Soldati, 2007;Koliwer-Brandl et al., 2019). Using this system, we previously found that after translocation into the host cytosol, M. marinum can escape from the cell by locally rupturing the plasma membrane, without host cell lysis (Gerstenmaier et al., 2015;Hagedorn et al., 2009). This egress occurs through a barrel-shaped F-actin structure, called the ejectosome. ...
... Strikingly, the tsg101-null mutant showed an almost complete lack of transmission at 24 hpi but recovered slightly at 32 F I G U R E 3 Quantitative comparison of bacterial transmission from atg1, tsg101, and double knock-out mutants. (a) a schematic representation of the quantitative transmission assay (modified from Hagedorn et al., 2009). Donor cells are infected with green fluorescent bacteria and at 6 hpi mixed with wild-type acceptor cells. ...
... Here, we present evidence that components of the ESCRT-complex also accumulate along the intracellular part of bacteria engaged in ejection. During ejection, bacteria breach the integrity of the host's plasma membrane on their way outwards, thereby introducing large damage or wounds to the cell (Hagedorn et al., 2009). Topologically, the ejectosome wound represents a special situation with the damaging force pushing against the membrane from the intracellular side. ...
Recruitment of both the ESCRT and autophagic machineries to ejecting Mycobacterium marinum
Article
Full-text available
  • May 2023
  • MOL MICROBIOL
Cytosolic Mycobacterium marinum are ejected from host cells such as macrophages or the amoeba Dictyostelium discoideum in a non‐lytic fashion. As described previously, the autophagic machinery is recruited to ejecting bacteria and supports host cell integrity during egress. Here, we show that the ESCRT machinery is also recruited to ejecting bacteria, partially dependent on an intact autophagic pathway. As such, the AAA‐ATPase Vps4 shows a distinct localization at the ejectosome structure in comparison to fluorescently tagged Vps32, Tsg101 and Alix. Along the bacterium engaged in ejection, ESCRT and the autophagic component Atg8 show partial colocalization. We hypothesize that both, the ESCRT and autophagic machinery localize to the bacterium as part of a membrane damage response, as well as part of a “frustrated autophagosome" that is unable to engulf the ejecting bacterium.
View
... After entry by phagocytosis in animal phagocytes of the innate immune system (8) or in amoebae (3,9), secretion of virulence factors, and especially of EsxA, via the ESX-1 secretion system inflicts membrane damage to the Mycobacteria-containing Vacuole (MCV) (10)(11)(12), which are repaired by a combination of cytosolic machinery. After cycles of damage and repair, the mycobacteria reach the cytosol where they continue to proliferate until they disseminate via lytic and non-lytic mechanisms (13)(14)(15)(16). ...
... To provide a comprehensive comparison and highlight evolutionary conservation, we conducted Tn-Seq experiments with M. marinum during infection of BV2 murine microglial cells. A single time point of 48 hpi was selected because, as in D. discoideum, a complete infection cycle in these phagocytes follows the same stages and also lasts approximately 48 hours (see Fig. 1A) (13,29,72). In total, seven samples were included in the analysis: four from the inoculum and three at 48 hpi (Table S2). ...
Temporal genome-wide fitness analysis of Mycobacterium marinum during infection reveals the genetic requirement for virulence and survival in amoebae and microglial cells
Article
Full-text available
  • Jan 2024
The emergence of biochemically and genetically tractable host model organisms for infection studies holds the promise to accelerate the pace of discoveries related to the evolution of innate immunity and the dissection of conserved mechanisms of cell-autonomous defenses. Here, we have used the genetically and biochemically tractable infection model system Dictyostelium discoideum / Mycobacterium marinum to apply a genome-wide transposon-sequencing experimental strategy to reveal comprehensively which mutations confer a fitness advantage or disadvantage during infection and compare these to a similar experiment performed using the murine microglial BV2 cells as host for M. marinum to identify conservation of virulence pathways between hosts.
View
... We and others have pioneered the investigation of various membrane repair pathways such as ESCRT-and autophagy- (López-Jiménez et al., 2018), as well as ER-dependent repair (Anand et al., 2023) In D. discoideum, light microscopy (LM) of live cells has been increasingly important to study the course of infection due to its high temporal and spatial resolution. For example, LM was used to generate time-lapse movies to monitor the escape of M. marinum via ejectosomes (Hagedorn et al., 2009), the spatiotemporal dynamics of Rab proteins at the MCV (Barisch, Lopez-Jimenez, & Soldati, 2015) and the re-distribution of lipid droplets during infection (Barisch, Paschke, et al., 2015). Although LM is a powerful tool for observing dynamic processes, it does have limitations. ...
... In the D. discoideum/M. marinum model system, TEM protocols including chemical fixation were used to image ejectosomes (Hagedorn et al., 2009) and to monitor accumulation of intracytosolic lipid inclusions inside M. marinum . However, conventional sample preparation for EM is known to induce artefacts such as cell shrinkage and extraction of cellular material, which can drastically alter the ultrastructure of the sample (McDonald & Auer, 2006). ...
Resolving exit strategies of mycobacteria in Dictyostelium discoideum by combining high-pressure freezing with 3D-correlative light and electron microscopy
Article
Full-text available
  • Dec 2023
  • MOL MICROBIOL
The infection course of Mycobacterium tuberculosis is highly dynamic and comprises sequential stages that require damaging and crossing of several membranes to enable the translocation of the bacteria into the cytosol or their escape from the host. Many important breakthroughs such as the restriction of mycobacteria by the autophagy pathway and the recruitment of sophisticated host repair machineries to the Mycobacterium‐containing vacuole have been gained in the Dictyostelium discoideum/M. marinum system. Despite the availability of well‐established light and advanced electron microscopy techniques in this system, a correlative approach integrating both methods with near‐native ultrastructural preservation is currently lacking. This is most likely due to the low ability of D. discoideum to adhere to surfaces, which results in cell loss even after fixation. To address this problem, we improved the adhesion of cells and developed a straightforward and convenient workflow for 3D‐correlative light and electron microscopy. This approach includes high‐pressure freezing, which is an excellent technique for preserving membranes. Thus, our method allows to monitor the ultrastructural aspects of vacuole escape which is of central importance for the survival and dissemination of bacterial pathogens.
View
... Despite being in an ILI depleted state 143 immediately upon infection (Fig. 1A), WT bacteria formed ILI throughout the course of the 72-144 hour infection, accumulating increasing levels of ILI until the point of host cell rupture (Fig. 1B [48]. In M. marinum, this perforation results in liberation of the bacteria into 153 the cytosol and actin-based motility for a fraction of the bacteria [49], [50]. The ESX-1 secretion 154 system is also required for phagosome maturation arrest, and ESX-1 mutants have increased 155 ...
Mycobacterial formation of intracellular lipid inclusions is a dynamic process associated with rapid replication
Preprint
  • Aug 2023
Intracellular lipid inclusions (ILI) are triacylglyceride rich organelles produced by mycobacteria thought to serve as energy reservoirs. It is believed that ILI are formed as a result of a dosR mediated transition from replicative growth to non-replicating persistence (NRP). ILI rich Mycobacterium tuberculosis (Mtb) bacilli have been reported during infection and in sputum, establishing their importance in Mtb pathogenesis. Studies conducted in mycobacteria such as Mycobacterium smegmatis, Mycobacterium abscessus, or lab Mtb strains have demonstrated ILI formation in the presence of hypoxic, nitric oxide, nutrient limitation, or low nitrogen stress, conditions believed to emulate the host environment within which Mtb resides. Here, we show that M. marinum and clinical Mtb isolates make ILI during active replication in axenic culture independent of environmental stressors. By tracking ILI formation dynamics we demonstrate that ILI are quickly formed in the presence of fresh media or exogenous fatty acids but are rapidly depleted while bacteria are still actively replicating. We also show that the cell envelope is an alternate site for neutral lipid accumulation observed during stationary phase. In addition, we screen a panel of 60 clinical isolates and observe variation in ILI production during early log phase growth between and among Mtb lineages. Finally, we show that dosR expression level does not strictly correlate with ILI accumulation in fresh clinical isolates. Taken together, our data provide evidence of an active ILI formation pathway in replicating mycobacteria cultured in the absence of stressors, suggesting a decoupling of ILI formation from NRP.
View
... Our results were in accordance with other reports that showed release of vesicles from protozoa that contained intracellular bacteria such as Escherichia coli O157 [23], Listeria monocytogenes [24] and Salmonella enterica [25]. Furthermore, release of free bacteria such as Legionella pneumophila [26] and mycobacteria [27] from intact amoeba host has been reported. Release of intracellular bacteria as free or encased in EVs have been regarded as peaceful strategies, compared with lytic escape of intracellular bacteria that destroys the host cell [28,29]. ...
Physicochemical properties of intact fungal cell wall determine vesicles release and nanoparticles internalization
Article
Full-text available
  • Feb 2023
Our previous microscopic observations on the wet mount of cultured Candida yeast showed release of large extracellular vesicles (EVs) that contained intracellular bacteria (∼500–5000 nm). We used Candida tropicalis, to examine the internalization of nanoparticles (NPs) with different properties to find out whether the size and flexibility of both EVs and cell wall pores play role in transport of large particles across the cell wall.Candida tropicalis was cultured in N-acetylglucoseamine-yeast extract broth (NYB) and examined for release of EVs every 12 h by the light microscope. The yeast was also cultured in NYB supplemented with of 0.1%, 0.01% of Fluorescein isothiocyanate (FITC)-labelled NPs; gold (0.508 mM/L and 0.051 mM/L) (45, 70 and 100 nm), albumin (0.0015 mM/L and 0.015 mM/L) (100 nm) and Fluospheres (0.2 and 0.02%) (1000 and 2000 nm). Internalization of NPs was recorded with fluorescence microscope after 30 s to 120 min. Release of EVs mostly occurred at 36 h and concentration of 0.1% was the best for internalization of NPs that occurred at 30 s after treatment. Positively charged 45 nm NPs internalized into >90% of yeasts but 100 nm gold NPs destroyed them. However, 70 nm gold and 100 nm negatively-charged albumin were internalized into
View
... These latter type of mechanism is generally called "vomocytosis", and it depends on the interplay between the pathogen and host-cell factors, such as actin polymerization, microtubule modulation, phagosomal pH, inflammation, and exocytosis signals, among others (1-3). Several mechanisms have been described for this type of "silent" egress, such as the ones utilized by Cryptococcus neoformans (31), Legionella pneumophila (32,33), and Mycobacterium tuberculosis (34). ...
Brucella Egresses from Host Cells Exploiting Multivesicular Bodies
Article
Full-text available
  • Jan 2023
Host cell egress is a critical step in the life cycle of intracellular pathogens, especially in microbes capable of establishing chronic infections. The Gram-negative bacterium Brucella belongs to such a group of pathogens. Even though much has been done to understand how Brucella avoids killing and multiplies in its intracellular niche, the mechanism that this bacterium deploys to egress from the cell to complete its cycle has been poorly studied. In the manuscript, we quantify the kinetics of bacterial egress and show that Brucella exploits multivesicular bodies to exit host cells. For the first time, we visualized the process of egress in real time by live video microscopy and showed that a population of intracellular bacteria exit from host cells in vacuoles containing multivesicular body-like features. We observed the colocalization of Brucella with two multivesicular markers, namely, CD63 and LBPA, both during the final stages of the intracellular life cycle and in egressed bacteria. Moreover, drugs that either promote or inhibit multivesicular bodies either increased or decreased the number of extracellular bacteria, respectively. Our results strongly suggest that Brucella hijacks multivesicular bodies to exit the host cells to initiate new infection events. IMPORTANCE How intracellular bacterial pathogens egress from host cells has been poorly studied. This is particularly important because this stage of the infectious cycle can have a strong impact on how the host resolves the infection. Brucella is an intracellular pathogen that infects mammals, including humans, and causes a chronic debilitating illness. The bacterium has evolved a plethora of mechanisms to invade host cells, avoid degradation in the endocytic pathway, and actively multiply within a specialized intracellular compartment. However, how this pathogen exits from infected cells to produce reinfection and complete its life cycle is poorly understood. In the manuscript, we shed some light on the mechanisms that are exploited by Brucella to egress from host cells. We observed for the first time the egress of Brucella from infected cells by time-lapse video microscopy, and we found that the bacterium exits in vesicles containing multivesicular bodies (MVBs) features. Moreover, the drug manipulation of MVBs resulted in the alteration of bacterial egress efficiency. Our results indicate that Brucella hijacks MVBs to exit host cells and that this strongly contributes to the reinfection cycle.
View
View
Immune modulation through secretory autophagy
Article
  • Jun 2023
  • J CELL BIOCHEM
Autophagy is a central mechanism of cellular homeostasis through the degradation of a wide range of cellular constituents. However, recent evidence suggests that autophagy actively provides information to neighboring cells via a process called secretory autophagy. Secretory autophagy couples the autophagy machinery to the secretion of cellular content via extracellular vesicles (EVs). EVs carry a variety of cargo, that reflect the pathophysiological state of the originating cells and have the potential to change the functional profile of recipient cells, to modulate cell biology. The immune system has evolved to maintain local and systemic homeostasis. It is able to sense a wide array of molecules signaling disturbed homeostasis, including EVs and their content. In this review, we explore the emerging concept of secretory autophagy as a means to communicate cellular, and in total tissue pathophysiological states to the immune system to initiate the restoration of tissue homeostasis. Understanding how autophagy mediates the secretion of immunogenic factors may hold great potential for therapeutic intervention.
View
A TRAF-like E3 ubiquitin ligase TrafE coordinates ESCRT and autophagy in endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum
Article
Full-text available
  • Apr 2023
  • eLife
Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. This severe stress is recognised and controlled by the endosomal sorting complex required for transport (ESCRT) and the autophagy machineries, which are recruited to damaged membranes to either repair or to remove membrane remnants. Yet, insight is limited about how damage is sensed and which effectors lead to extensive tagging of the damaged organelles with signals, such as K63-polyubiquitin, required for the recruitment of membrane repair or removal machineries. To explore the key factors responsible for detection and marking of damaged compartments, we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase, TrafE, that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical compounds. TrafE acts at the intersection of ESCRT and autophagy pathways and plays a key role in functional recruitment of the ESCRT subunits ALIX, Vps32 and Vps4 to damage sites. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of mycobacteria as well as ESCRT-mediated and autophagy-mediated endolysosomal membrane damage repair, resulting in early cell death.
View
View
Differential Growth Characteristics and Streptomycin Susceptibility of Virulent and Avirulent Mycobacterium tuberculosis Strains in a Novel Fibroblast-Mycobacterium Microcolony Assay
Article
Full-text available
  • Dec 1998
The ability to spread from cell to cell may be an important virulence determinant of Mycobacterium tuberculosis. An in vitro assay was developed to characterize this ability among four strains of M. tuberculosis: the attenuated strain H37Ra, the virulent strains H37Rv and Erdman, and a virulent clinical isolate (Stew). Confluent monolayers of human skin fibroblasts were infected with these strains and overlaid with agar-medium. M. tuberculosis infection developed over 21 days as microcolonies originating within the plane of the fibroblasts. Microcolonies of the virulent strains had an elongated appearance and exhibited extensive cording. The cords appeared to invade adjacent cells within the plane of the monolayer. Microcolony diameter of the Erdman strain was significantly larger than that of the other virulent strains, indicating that virulent strains can have distinguishing phenotypes in this assay. In contrast, avirulent H37Ra microcolonies were rounded and noncorded. H37Ra microcolonies were significantly smaller than those of the virulent strains. Microcolony diameter of the virulent strains was not reduced by the extracellularly acting antibiotic streptomycin at concentrations of up to 5.0 microgram/ml. In contrast, H37Ra microcolony size was reduced at concentrations as low as 0.5 microgram/ml. Growth of all strains was similarly inhibited by 1.0 microgram of streptomycin per ml in fibroblast-conditioned tissue culture medium alone. When fibroblasts were infected with the M. tuberculosis strains without an agar overlay, with and without streptomycin, numbers of CFU mirrored the changes observed in the microcolony assay. There was a statistically significant decrease in H37Ra CFU compared to virulent strains after treatment with streptomycin. These differences between H37Ra and virulent strains in human fibroblasts suggest that H37Ra may be lacking a virulence determinant involved in cell-to-cell spread of M. tuberculosis.
View
Contraction and polymerization cooperate to assemble and close actomyosin rings around Xenopus oocyte wounds
Article
Full-text available
  • Sep 2001
Xenopus oocytes assemble an array of F-actin and myosin 2 around plasma membrane wounds. We analyzed this process in living oocytes using confocal time-lapse (four-dimensional) microscopy. Closure of wounds requires assembly and contraction of a classic "contractile ring" composed of F-actin and myosin 2. However, this ring works in concert with a 5-10-microm wide "zone" of localized actin and myosin 2 assembly. The zone forms before the ring and can be uncoupled from the ring by inhibition of cortical flow and contractility. However, contractility and the contractile ring are required for the stability and forward movement of the zone, as revealed by changes in zone dynamics after disruption of contractility and flow, or experimentally induced breakage of the contractile ring. We conclude that wound-induced contractile arrays are provided with their characteristic flexibility, speed, and strength by the combined input of two distinct components: a highly dynamic zone in which myosin 2 and actin preferentially assemble, and a stable contractile actomyosin ring.
View
The Secret Lives of the Pathogenic Mycobacteria
Article
Full-text available
  • Feb 2003
Pathogenic mycobacteria, including the causative agents of tuberculosis and leprosy, are responsible for considerable morbidity and mortality worldwide. A hallmark of these pathogens is their tendency to establish chronic infections that produce similar pathologies in a variety of hosts. During infection, mycobacteria reside in macrophages and induce the formation of granulomas, organized immune complexes of differentiated macrophages, lymphocytes, and other cells. This review summarizes our understanding of Mycobacterium-host cell interactions, the bacterial-granuloma interface, and mechanisms of bacterial virulence and persistence. In addition, we highlight current controversies and unanswered questions in these areas.
View
Mycobacterium marinum Escapes from Phagosomes and Is Propelled by Actin-based Motility
Article
Full-text available
  • Dec 2003
Mycobacteria are responsible for a number of human and animal diseases and are classical intracellular pathogens, living inside macrophages rather than as free-living organisms during infection. Numerous intracellular pathogens, including Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsii, exploit the host cytoskeleton by using actin-based motility for cell to cell spread during infection. Here we show that Mycobacterium marinum, a natural pathogen of fish and frogs and an occasional pathogen of humans, is capable of actively inducing actin polymerization within macrophages. M. marinum that polymerized actin were free in the cytoplasm and propelled by actin-based motility into adjacent cells. Immunofluorescence demonstrated the presence of host cytoskeletal proteins, including the Arp2/3 complex and vasodilator-stimulated phosphoprotein, throughout the actin tails. In contrast, Wiskott-Aldrich syndrome protein localized exclusively at the actin-polymerizing pole of M. marinum. These findings show that M. marinum can escape into the cytoplasm of infected macrophages, where it can recruit host cell cytoskeletal factors to induce actin polymerization leading to direct cell to cell spread.
View
Demonstration of spread by Mycobacterium tuberculosis bacilli in A549 epithelial cell monolayers
Article
  • Jul 2002
  • FEMS MICROBIOL LETT
We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.
View
The Role of the Granuloma in Expansion and Dissemination of Early Tuberculous Infection
Article
  • Feb 2009
Granulomas, organized aggregates of immune cells, form in response to persistent stimuli and are hallmarks of tuberculosis. Tuberculous granulomas have long been considered host-protective structures formed to contain infection. However, work in zebrafish infected with Mycobacterium marinum suggests that granulomas contribute to early bacterial growth. Here we use quantitative intravital microscopy to reveal distinct steps of granuloma formation and assess their consequence for infection. Intracellular mycobacteria use the ESX-1/RD1 virulence locus to induce recruitment of new macrophages to, and their rapid movement within, nascent granulomas. This motility enables multiple arriving macrophages to efficiently find and phagocytose infected macrophages undergoing apoptosis, leading to rapid, iterative expansion of infected macrophages and thereby bacterial numbers. The primary granuloma then seeds secondary granulomas via egress of infected macrophages. Our direct observations provide insight into how pathogenic mycobacteria exploit the granuloma during the innate immune phase for local expansion and systemic dissemination.
View
Recent quantitative studies of actin filament turnover during cell locomotion
Article
  • Jan 1993
Cell locomotion depends on polymerization and depolymerization of filamentous actin. Net polymerization at the cell front occurs fast enough to fill the extending lamellipod, and since total F-actin is essentially constant over time, depolymerization must equal polymerization. Indeed, the fastest moving cell types have the highest rates of depolymerization. Accounting for the high rate of depolymerization raises several problems. One is that net depolymerization requires the concentration of G-actin to be low (below the critical concentration), but rapid polymerization (occurring < 1 micron away) requires the concentration of G-actin to be high (well above the critical concentration). This may be accomplished by spatial compartmentalization of factors that favor polymerization or depolymerization, and/or by proteins that bind G-actin and prevent spontaneous polymerization while allowing barbed-end elongation. A second problem is that depolymerization proceeds faster than would seem possible from studies of F-actin in vitro (as calculated from number and lengths of filaments present and in vitro rate constants). Rapid depolymerization may be accomplished by filament cutters or by cytoplasmic components (as yet undiscovered) that increase the rate of depolymerization.
View
Visualization of Actin Dynamics During Macropinocytosis and Exocytosis
Article
  • Apr 2002
Macropinocytosis of newly formed resides and exocytosis of post-lysosomes have been visualized using a green fluorescent protein probe that binds specifically to F-actin filaments. F-actin association with macropinocytosis begins as a V-shaped infolding of the membrane. Vesicle enlargement occurs through an inward movement of the proximal point of the V as well as an outward protrusion at the tip of the V to form an elongated invagination. The protrusion eventually closes at its distal margin to become a vesicle and is moved centripetally while recovering its circular shape. The vesicle loses its actin coat within 1 min after internalization. One hour later, post-lysosomal vesicles became weakly surrounded by actin while still cytoplasmic. Some of these vesicles moved to the plasma membrane, docked, and then expelled their contents. Slightly before the vesicle content began to disappear, an increase in F-actin association with the vesicle was observed. This was followed by rapid contraction of the vesicle and then disappearance of the actin signal once the internal content was released. These results show that dynamic changes in actin filament association with the vesicle membrane accompany both endocytosis and exocytosis.
View
Demonstration of spread by Mycobacterium tuberculosis bacilli in A549 epithelial cell monolayers
Article
  • Aug 2002
We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.
View
Real-Time Visualization of Mycobacterium-Macrophage Interactions Leading to Initiation of Granuloma Formation in Zebrafish Embryos
Article
  • Jan 2003
  • IMMUNITY
Infection of vertebrate hosts with pathogenic Mycobacteria, the agents of tuberculosis, produces granulomas, highly organized structures containing differentiated macrophages and lymphocytes, that sequester the pathogen. Adult zebrafish are naturally susceptible to tuberculosis caused by Mycobacterium marinum. Here, we exploit the optical transparency of zebrafish embryos to image the events of M. marinum infection in vivo. Despite the fact that the embryos do not yet have lymphocytes, infection leads to the formation of macrophage aggregates with pathological hallmarks of granulomas and activation of previously identified granuloma-specific Mycobacterium genes. Thus, Mycobacterium-macrophage interactions can initiate granuloma formation solely in the context of innate immunity. Strikingly, infection can redirect normal embryonic macrophage migration, even recruiting macrophages seemingly committed to their developmentally dictated tissue sites.
View