Superinfecting mycobacteria home to established granulomas

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Superinfecting mycobacteria home to established
granulomas

• Christine L. Cosma, Oliver Humbert Lalita
  Ramakrishnan
• Presented by Donica Larson
• May 10, 2005

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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

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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 gt 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

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Electron micrograph of Mycobacterium tuberculosis
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Lung granuloma
? Caseous necrosis ?
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Colonies of Mycobacterium tuberculosis on
Lowenstein-Jensen medium. CDC
Acid Fast stain
Mycobacterium
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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

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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

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Results

• 1) Protection against M. marinum superinfection
  in preinfected frogs

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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 pre-infected frogs

Result Immunity to mycobacterial infections is
functionally similar in frogs and mammals
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2) Mycobacteria transport to established
granulomas via host cells
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Where are superinfecting bacteria localized with
respect to previously established granulomas?
Figure 2

• Arrow epithelioid macrophages

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• 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
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Figure 3
Figure 2

• The superinfecting strain also established its
  own granulomas.

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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

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Figure 4
a) Infected w/ red, s.i. gt7 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
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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

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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.
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3) Mycobacterium-infected monocytes show
enhanced trafficking
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Is the accumulation of mycobacterium-infected
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

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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 5
Figure 4
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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.
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4) Trafficking is pathogen specific
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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

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• S. arizonae-infected cells located in many small
  aggregates _at_ 3 days and in fewer larger
  aggregates _at_ 7 days

M. marinum red S. arizonae green
Figure 6
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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
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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
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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)
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5) Superinfecting bacteria penetrate caseum of
mature granulomas
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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

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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.
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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.
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6) Rapid induction of granuloma-specific genes
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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

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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
  gag7gfp 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
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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

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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

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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

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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

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References

• http//en.wikipedia.org/wiki/Tuberculosis
• http//www.sanger.ac.uk/Projects/M_tuberculosis/
• http//dermatlas.med.jhmi.edu/derm/result.cfm?Diag
  nosis1494087972
• http//www.textbookofbacteriology.net/tuberculosis
  .html
• http//www-medlib.med.utah.edu/WebPath/TUTORIAL/MT
  B/MTB.html
• http//www.sciencedaily.com/releases/2004/10/04103
  0154143.htm

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Happy 2nd Birthday, Breanna!