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

↑ Caseous necrosis → Lung granuloma

Colonies of Mycobacterium tuberculosis on Lowenstein-Jensen medium. CDC Acid Fast stain Mycobacterium

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 10 6 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

2) Mycobacteria transport to established granulomas via host cells

Arrow: epithelioid macrophages Where are superinfecting bacteria localized with respect to previously established granulomas? Figure 2

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

The superinfecting strain also established its own granulomas. Figure 2 Figure 3

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

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 Figure 4

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… and outside preexisting granulomas Therefore, the bacteria are intracellular and the entry of superinfecting bacteria into granulomas is accomplished by migration of infected host cells. Fig. 4

3) Mycobacterium-infected monocytes show enhanced trafficking

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

Results: Similar proportions of bacteria and beads reached the liver 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 5Figure 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 Results: Greater proportion of beads in granulomas when coinjected. Enhanced entry due to ↑ accumulation of infected monocytes, not preferential replication. Figure 5

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 3 days and in fewer larger 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 Result: There is distinct localization of the 2 pathogens with respect to established granulomas (pathogen-specific effect) Figure 6

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. Superinfection w/ green fluorescent beads Beads reached caseum by 11 d.p.i Figure 7

Is the caseum penetrated? Mycobacteria (green) reached cellular layer surrounding caseum by 2 h.p.i. Entered the caseum by 5 d.p.i. Figure 7

Superinfecting bacteria formed their own granulomas by 5 d.p.i., but had not yet developed caseous centers Result: Uninfected and mycobacterium-infected cells rapidly penetrate caseous granulomas, including the caseum. Figure 7

6) Rapid induction of granuloma- specific 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 medlib.med.utah.edu/WebPath/TUTORIAL/MTB/MTB.html

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