Transcript Superinfecting mycobacteria home to established granulomas

Superinfecting mycobacteria
home to established granulomas
Christine L. Cosma, Oliver Humbert & Lalita
Ramakrishnan
Presented by Donica Larson
May 10, 2005
Background
• Mycobacterium tuberculosis
– Gram-positive rod
• Non-motile
– Acid Fast positive
– Causative agent of tuberculosis (TB)
– Often is in state of clinical latency
• Bacteria persist undetected for decades before leading to
active disease
Background
• Tuberculosis is the most common major infectious
disease today, infecting two billion people.
– 9 million new cases of active disease annually
– 2 million deaths, mostly in developing countries
• 90% have asymptomatic latent TB infection (LTBI).
– 10% lifetime chance that LTBI will progress to active TB disease
– untreated, kills > 50% of victims
– One of the top three infectious killing diseases in the world
• HIV/AIDS kills 3 million people each year
• TB kills 2 million
• malaria kills 1 million
Electron micrograph of Mycobacterium tuberculosis
Lung granuloma
↑
Caseous necrosis
→
Colonies of Mycobacterium tuberculosis on Lowenstein-Jensen medium. CDC
Mycobacterium
Acid Fast stain
Introduction
• The bacteria
– Mycobacterium marinum: causes systemic chronic tuberculosis
in ectotherms
– Salmonella enterica serovar Arizonae: macrophage pathogen of
ectotherms
• The hosts
– Frog model (Rana pipiens): study of trafficking into granulomas
– Zebrafish model (Danio rerio): observation of caseating
granulomas
• Neither is demonstrated in mouse model
Introduction
• Infection: initial infection causing granulomas
• Superinfection: subsequent infection after
granulomas are established
• Granuloma: complex immune structures that
comprise differentiated macrophages,
lymphocytes & other immune cells
• Caseum: located w/in granulomas, central area
of necrosis. Develop in mature lesions
Results
1) Protection against M. marinum
superinfection in preinfected frogs
Do frogs chronically infected w/ M. marinum
have partial immunity to superinfections?
• Pre-infect frogs w/ wt M. marinum
for 6 wks (3.3 x 106 CFU)
• Infect naïve and pre-infected frogs
w/ kanamycin resistant
M. marinum
• Count kanamycin resistant
bacteria 2 wks and 8 wks later
• 1-2 log less burden in preinfected frogs
Result: Immunity to mycobacterial infections is functionally
similar in frogs and mammals
2) Mycobacteria transport to
established granulomas via host cells
Where are superinfecting bacteria localized with
respect to previously established granulomas?
Figure 2
Arrow: epithelioid macrophages
•Infect frogs w/ red
fluorescent M. marinum for
8 wks to establish chronic
granulomatous infection
•Superinfect frogs w/ green
fluorescent bacteria
•Examine tissue at 10 wks
and 20 wks after
superinfection
Result: superinfecting strain
penetrated readily and
persisted in previously
established granulomas
Figure 2
Figure 3
Figure 2
•The superinfecting strain also established its own granulomas.
Do the mixed granulomas represent trafficking of
superinfecting bacteria into established
granulomas, or remodeling of adjacent granulomas
first established separately by the 2 strains?
• Superinfect frogs as described previously
– red = initial infection
– green = superinfection
• Examine localization of superinfecting strain at 3 days
p.i.
• Count individual green fluorescent bacteria
Figure 4
a) Infected w/ red, s.i. >7 wks w/ green
b,c) Infected w/ cyan, s.i.
double-labeled bacteria (green
& red = yellow)
d) Infected w/ red M. marinum, s.i.
green M. marinum
Are superinfecting mycobacteria gaining
passage into established granulomas w/in
host cells?
• Used red fluorescent strain of M. marinum that
expresses GFP from a macrophage-activated promoter,
induced by entry into host cells
• All bacteria are red, but only those located intracellularly
are green
• Superinfect frogs w/ this doubly marked strain into frog
previously infected with cyan fluorescent M. marinum
Result: At 3 days post superinfection, dually red/green fluorescent
bacteria were within preexisting granulomas…
Fig. 4
and outside preexisting granulomas
Fig. 4
Therefore, the bacteria are intracellular and the entry of superinfecting
bacteria into granulomas is accomplished by migration of infected host cells.
3) Mycobacterium-infected
monocytes show enhanced
trafficking
Is the accumulation of mycobacteriuminfected cells in granulomas enhanced
compared with that of uninfected phagocytic
cells?
• Compared rates of accumulation of M. marinum
vs. immunologically inert green latex beads
• Injected green M. marinum or beads into frogs
w/ established red M. marinum infections
• Counted bacteria and beads in pre-established
liver granulomas after 3 days
Results: Similar proportions of bacteria and beads reached
the liver
Figure 5
While a few beads were present in the preexisting granulomas,
the proportion of M. marinum trafficking to preexisting
granulomas was 4- to 5-fold greater than that of the beads
Figure 4
Figure 5
Is the greater number of superinfecting bacteria
compared with the beads in pre-established
lesions due to preferential replication, rather than
increased trafficking?
• Compared the rate of entry of beads coinjected with
M. marinum w/ that of beads alone
Figure 5
Results: Greater proportion of beads in granulomas when
coinjected. Enhanced entry due to ↑ accumulation of infected
monocytes, not preferential replication.
4) Trafficking is pathogen specific
Is the trafficking of mycobacterium-infected
cells into granulomas pathogen specific?
• Examined localization of Salmonella enterica
serovar Arizonae (S. arizonae) with respect to
established granulomas in M. marinum-infected
frogs
• S. arizonae-infected cells located in many small
aggregates @ 3 days and in fewer larger
aggregates @ 7 days
M. marinum = red
S. arizonae = green
Figure 6
A few S. arizonae were present in preexisting M. marinum
granulomas at 3 days
Similar results were obtained when comparing with beads
(previous experiment)
Figure 5
Is the trafficking pathogen specific in
competition?
• Frogs had established M. marinum granulomas
• Frogs were superinfected w/ M. marinum and
S. arizonae simultaneously
Result: Higher proportion of granulomas had M. marinum despite similar bacterial loads
The close juxtaposition of the large Salmonella granulomas and
smaller Mycobacterium granulomas …
did not result in co-mingling
Figure 6
Result: There is distinct localization of the
2 pathogens with respect to established
granulomas (pathogen-specific effect)
5) Superinfecting bacteria penetrate
caseum of mature granulomas
Do superinfecting bacteria traffic to caseous
granulomas?
• Zebrafish infected w/ red fluorescent M. marinum
– Developed caseated granulomas by 6 wks p.i.
Figure 7
•Superinfection w/ green fluorescent beads
•Beads reached caseum by 11 d.p.i
Is the caseum penetrated?
Mycobacteria (green) reached cellular layer surrounding
caseum by 2 h.p.i.
Figure 7
Entered the caseum by 5 d.p.i.
Superinfecting bacteria formed their own
granulomas by 5 d.p.i., but had not yet developed
caseous centers
Figure 7
Result: Uninfected and mycobacterium-infected
cells rapidly penetrate caseous granulomas,
including the caseum.
6) Rapid induction of granulomaspecific genes
How do superinfecting mycobacteria that rapidly
enter mature granulomas adapt to survive the
established host immune response?
• Stepwise adaptation during primary infection
• macrophage-activated genes (mag genes)
expressed after entry into host macrophages
• granuloma-activated genes (gag genes) expressed
after macrophage aggregation into granulomas
Does superinfecting M. marinum express
gag genes immediately after entering
mature granulomas?
• Used frogs w/ granulomas established by red
fluor. bacteria
• Superinfected frogs w/ M. marinum bearing
gag7:gfp fusion
• Examined 3 d.p.i. for green bacteria
Result: gag expression is
rapidly induced when individual
bacteria enter established
granulomas (rapidly adapt to
granuloma environment).
Fig. 8
Discussion
• There is enhanced migration to granulomas of
mycobacterium-infected cells compared with that
of uninfected phagocytes.
– Tendency of mycobacterium-infected cells to
aggregate into new lesions
• Established granulomas compete effectively for
mycobacterium-infected monocytes
Discussion
• Superinfecting mycobacteria were rapidly
transported to caseating granulomas by host
mononuclear cells
• Salmonella-infected cells were excluded from
mycobacterium granulomas
– Mononuclear cells infected by different pathogens
pursue distinct trafficking patterns
• Granulomas fail to eradicate superinfecting
bacteria
Discussion
• Suggest the caseum produces signals to attract
infected and uninfected monocytes
• The caseum is not physically walled off from the
surrounding tissue
• Mycobacterial adaptation mechanisms initiate
rapidly to ensure survival in a mature granuloma
environment
Current and Future studies
• Infection of M. marinum with the transparent
zebrafish embryos enabled Ramakrishnan and
colleagues to observe cells recruited to
granulomas
– Findings: RD1 (a genetic region involved in virulence)
is required for granuloma formation and even recruits
more macrophages to granulomas
• Developing markers to distinguish macrophages
and dendritic cells in zebrafish
References
• http://en.wikipedia.org/wiki/Tuberculosis
• http://www.sanger.ac.uk/Projects/M_tuberculosis/
• http://dermatlas.med.jhmi.edu/derm/result.cfm?Diagnosis=14940879
72
• http://www.textbookofbacteriology.net/tuberculosis.html
• http://wwwmedlib.med.utah.edu/WebPath/TUTORIAL/MTB/MTB.html
• http://www.sciencedaily.com/releases/2004/10/041030154143.htm
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