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大家好
-Hello.

我是Roland Brosh
This is Roland Brosh.

我是分枝杆菌病原基因组学
I am the head

研究组的负责人
of the Unit for Integrated Mycobacterial Pathogenomics

也是巴斯德研究院的教授
and a professor at the Pasteur Institute.

我们致力于结核分枝杆菌
We work on Mycobacterium tuberculosis

和其他导致结核病的分枝杆菌
and other tuberculosis-causing mycobacteria.

在生命之树中
In the tree of life,

引起结核的分枝杆菌属于放线菌门
tuberculosis-causing mycobacteria belong to the genus Actinobacteria.

不同于厚壁菌和变形菌
They are different from the Firmicutes and also from the Proteobacteria.

厚壁菌通常含有革兰氏阳性菌
The Firmicutes normally contain the gram-positive bacteria

而变形菌含有革兰氏阴性菌
and the Proteobacteria contain the gram-negative bacteria.

放线菌是非常不同的类别
Actinobacteria are really a different class

分枝杆菌也一样
so are mycobacteria.

其中有生长速度快和速度慢的两种类型
Among them we have fast growers and slow growers.

130多种分枝杆菌中的大多数是非致病
Most of the species, most of the more than 130 species of mycobacteria

生长速度快的类型除去一个例外
are non-pathogenic, fast growers, with one exception,

脓肿分枝杆菌
Mycobacterium abscessus

是囊胞性纤维症病人的病原体
which is a pathogen for cystic fibrosis patients.

在16S RNA树中有一组生长缓慢的细菌
Then, we have one group in the 16S RNA tree which are the slow-growing bacteria.

这之中有主要病原体
Among them we have the major pathogens.

例如 这里有鱼类和人类的病原体
Here we have for instance the fish and human pathogen,

海分枝杆菌
Mycobacterium marinum.

溃疡分枝杆菌
We have Mycobacterium ulcerans

是布鲁里溃疡的病原体
which is the agent of Buruli ulcer.

麻风分枝杆菌
We have the agent of leprosy,

麻风病的病原体
Mycobacterium leprae.

堪萨斯分枝杆菌
We have Mycobacterium kansasil

这是一种人类机会性病原体
which is a human opportunistic pathogen.

还有动物约尼氏病的病原体
We also have an animal pathogen of Johne's disease,

鸟的副结核分枝杆菌
Mycobacterium avium paratuberculosis.

当然 还有结核病的病原体
And of course we have the etiological agent of tuberculosis.

结核分枝杆菌复合体
This is the M. tuberculosis complex.

这种复合体的个体
Members of this complex

是能导致不同哺乳动物结核病的病原体
can cause tuberculosis in different mammalian species.

从这些结核分枝杆菌的演进过程中
What do we know about the evolution from these tuberculosis

我们能知道什么
-causing mycobacteria?

他们共同的祖先传播到不同的种群
Their common ancestor spread into different lineages

有的进化为牛分枝杆菌
and we have here one lineage which goes towards Mycobacterium bovis

这是一种动物病原体
which is an animal pathogen.

第一个遗传事件是区域9的缺失
The first genetic event was the deletion of the region 9.

也就是基因缺失
So, it is a genetic deletion.

这里的所有菌株都是非洲分枝杆菌
All of these strains here, Mycobacterium africanum,

是西非人群中的一种病原体
which is a pathogen in humans in West Africa,

有2种不同的谱系
2 different lineages,

它们的RD9缺失
they are RD9-deleted.

鼠分枝杆菌是公牛的病原体
Mycobacterium microti which is a pathogens for bulls.

M. pinnipedii是海豹的病原体
M. pinnipedii for seals,

M. oryx是羚羊的病原体
M. oryx for antelopes,

M. caprae是山羊的病原体
M. caprae for goats,

之后是典型的牛分枝杆菌菌株
and then the classical M. bovis strains,

再往后是来自牛分枝杆菌的卡介苗菌株
and then the BCG strains which were derived from M. bovis.

正如我所说 我们有三个主要的谱系
We have three main lineages as I said.

第一个是具有完整的TbD1的
The first one is TbD1 intact.

这些是古代结核分枝杆菌菌株
So, these are the ancestral Mycobacterium tuberculosis strains.

第二个是删除了TbD1的
The second lineage is TbD1 deleted.

这些是所谓的 现代 结核分枝杆菌菌株
These are the so-called "modern" M. tuberculosis strains.

它们都是人类的病原体
Both of them are of course human pathogens.

还有RD9缺失的谱系
Then, we have the RD9-deleted lineage,

包含非洲菌株和动物菌株
like I explained, containing the M. africanum strains and then the animal strains.

我们有无性系种群结构
We have a clonal population structure

因为没有在不同菌株之间的进行缺失交换
because we do not have an exchange of deletions among the different strains.

对于这里展示的一些病原体
We have attenuation which was obtained, for some of the members shown here,

RD1缺失减弱了鼠分枝杆菌的毒性
by the RD1 deletion in Mycobacterium microti.

在同样的区域不同的RD1缺失
A different RD1 deletion but in the same region

也会缺少ESX-1分泌系统
which also took away the ESX-1 secretion system

导致卡介苗毒性减弱
caused the attenuation of BCG.

可以从这张幻灯片中得出结论
We can also conclude from this slide that M.bovis

牛结核杆菌
is not the progenitor

不是结核分枝杆菌（Mtb）的祖先
of Mtb

因为它来自这个单独谱系
because it comes in this separate lineage.

坎纳分枝杆菌
Mycobacterium canettii

是一个像这里一样的早期分支群
is an early branching outgroup like here.

我之后会谈论这一点
I will speak about this in a second.

NGS第二代测序技术
NGS, Next-Generation Sequencing,

通过分析缺失确定了这一点
confirmed this scheme identified by deletion analyses.

这是Bos等人
This is a representation

发表在《Nature》上的一篇文章
which was published by Bos et al.

很好的展示了不同谱系
in "Nature" which nicely shows the different lineages.

这里是谱系1人类菌株
We have lineage 1, human strains,

始祖结核分枝杆菌菌株
ancestor Mtb strains.

然后就像我说的
Then, like I said,

RD9缺失的谱系5和谱系6
lineage 5, lineage 6 here with the RD9 deletion,

也是人类的菌株
also human strains

从这里到动物菌株进化而来的
from here to animal strains evolved.

有一个最近确定的谱系7
We have a lineage 7 which was recently identified.

现代结核菌株是谱系2 3和4
Then, we have the modern TB strains which are lineage 4, 2 and 3.

共有7个谱系在人类中流行
We have in total 7 lineages prevalent in humans.

其中三个是 现代 的
Three of them are "modern"

TbD1缺失的菌株
and TbD1 deleted like for instance the L2 Beijing strains

例如现在流行在世界各地的L2北京株
which are prevalent now all around the world.

我们有从RD9缺失的M. africanum分支的
We have the animal strains, lineage 8,

动物菌株谱系8
which was branching from RD9-deleted M. africanum.

最多有2 500个单核苷酸多态位点（SNP）
There are 2 500 SNPs maximum.

这是一个无性系种群结构
It is a clonal population structure.

坎纳分枝杆菌是外群
M. canettii is the outgroup,

就像这里显示的那样
like shown here.

还有什么关于坎纳分枝杆菌的呢
What else do we know about M. canettii?

20世纪60年代
George Canetti

乔治•卡内蒂（George Canetti）
was the first one who isolated an Mtb-like strain

在巴斯德研究院第一次分离出
with smooth colony morphology here

平滑菌落形态的类似结核分枝杆菌的菌株
at the Pasteur Institute in the 1960s.

所以被称为坎纳分枝杆菌
Therefore they were called M. canettii.

Dick van Sooligen对此进行了定义
It was Dick van Sooligen who defined this.

从病人身上分离的菌株不到100种
There are less than 100 strains isolated from patients,

主要是在非洲之角的吉布提
mainly in Djibouti, the Horn of Africa.

基因组分析显示结核分枝杆菌
Genome analyses showed an important genomic diversity

和坎纳分枝杆菌之间有明显的基因组多样性
between M.tuberculosis and M. canettii strains,

而且在不同的坎纳分枝杆菌菌株之间
but also among different M. canettii strains,

也有16 000到65 000个SNP
16 000 to 65 000 SNPs in this group.

基因组分析也显示坎纳分枝杆菌菌株
Genome analyses also showed that M. canettii strains

是重组的
are recombinogenic.

毒性分析表明
Virulence analyses showed that M.canettii strains

与结核分枝杆菌相比
are less virulent in mouse infection models

小鼠感染模型中的坎纳菌株毒性较小
than Mtb.

所以先祖的毒性较弱
So, the progenitor is less virulent.

这张幻灯片告诉你坎纳分枝杆菌
This slide shows you that M.Canettii

可以有两种形式
can have two forms.

通常它很光滑
Normally it is smooth

但也有一些粗糙的菌落
but there are also some rough colonies.

你可以看到它们在液体培养基中聚团生长
You can see that they clamp in liquid culture.

在显微镜下也可以看到
This is also seen here under the microscope.

结核分枝杆菌
M. tuberculosis normally always

通常具有这种粗糙的菌落形态
N. has this rough colony morphology.

2016年发表在《自然微生物学》上的
This work published in "Nature Microbiology"

这篇文章写道
in 2016 showed that

实际上这种粗糙的菌落形态
in fact this rough colony morphology

是由于两个聚酮合酶基因的丢失
was caused by the loss, or the recombination,

或重组以及中间一个基因的缺失造成的
of two polyketide synthase genes and the deletion of one gene in the middle.

这两个基因重新组合
These two genes recombine

失去了产生脂寡糖的功能
and the function of lipo-oligosaccharide production was lost.

光滑菌落形态的消失
The loss of smooth colony morphology

是由于缺少脂寡糖的产生
is due to the lost lipo-oligosaccharide production.

这篇文章也可以证明粗糙的菌株
In this work we could also show that the rough strains

在豚鼠模型中毒性更强
were more virulent in the guinea pig model.

LOS缺失也与毒性增加有关
So the loss of LOS was also linked to a gain of virulence.

我们可以在这棵树上看到
We can see this on this tree.

这里有一群基因多样性强的
We have here the group of M.canettii strains

坎纳分枝杆菌菌株
which are genetically very diverse.

这种无性系种群
Then, we have this clonal population which arose,

显然是来自坎纳分枝杆菌类的祖先之一
apparently, from one of the M. canettii-like ancestors

成为结核分枝杆菌复合体
and became the M.tuberculosis

具有很少的多样性
complex with very little diversity

但具有很大的地理和宿主物种变异性
but a large geographic and host species variation.

可以看到
We also could see

当LOS消失时
that there was a change in the immune signaling

免疫信号也消失了
which was also lost when the LOS production disappeared.

坎纳分枝杆菌菌株可以重组
Mycobacterium canettii strains can recombine.

这可以在基因组中看到
This can be seen in the genome

也可以通过在这里使用不同的标记
and we also could show this experimentally

展示这个实验
by using different markers here.

例如
For instance,

绿色荧光标记和红色荧光标记
a green fluorescent marker and a red fluorescent marker.

可以证明一个供体菌株
We could show that a donor strain

能将大的DNA片段转移到受体菌株中
can transfer large DNA fragments into a recipient strain.

在这里
This is shown here.

转导结合体或重组体呈橙色
The transconjugant or the recombinant is in orange color

并具有两种不同的抗生素抗性基因盒
and has two different antibiotic resistance cassettes.

这只是PCR的确认
This is just the confirmation by PCR.

这是一张更复杂的幻灯片
This is a more complicated slide

告诉你其实
which should show you that in fact...

供体菌株显示在底部
The donor strain is shown here at the bottom.

受体菌株显示在顶部
The recipient strain is shown here at the top.

每条红线是一个SNP
Each red line is one of the single nucleotide polymorphisms.

中间的转接合子
The transconjugant in the middle

通常具有受体菌株的遗传相似性
has normally the genetic constellation of the recipient strain

但是在一些区域
but in some areas,

可以看到它已经变得像供体菌株
you can see that it has become like the donor strain.

放大看是这样
This is shown here in magnification.

这很好地证明了
This nicely shows that

DNA从一个菌株转移到另一个菌株
DNA was transferred from one strain to another.

有两个坎纳分枝杆菌菌株之间
There is experimental evidence of transfer of multiple,

转移多个大染色体片段的实验证据
large chromosomal fragments between two M. canettii strains.

我们也试图用结核分枝杆菌这样做
We also tried to do this

尽管进行了大量的试验
with M. tuberculosis

但没有成功
but did not succeed despite extensive trials.

这说明了结核分枝杆菌复合菌株的
This argues for a clonal population structure of the M.tuberculosis

无性系种群结构
complex strains,

这在以前的幻灯片中也可以看到
which is also seen in the slide before.

最后我来总结一下
This brings me to the conclusions.

引起结核病的分枝杆菌的发展
The evolution of tuberculosis-causing mycobacteria

从类似坎纳分枝杆菌的分枝杆菌开始
has started from an M. canettii-like pool of mycobacteria.

在演进过程中
During the recent evolution,

这个谱系失去了产生脂寡糖的能力
this lineage has lost the ability to produce lipo-oligosaccharides.

似乎也丧失了
During the recent evolution,

菌株间DNA转移的能力
this lineage also seems to have lost the ability for inter-strain transfer of DNA.

因此它具有无性系种群结构
Therefore, it has a clonal population structure.

正如我在缺失分析的幻灯片中显示的那样
The evolution of this lineage

现在被称为结核分枝杆菌复合体的
which is now called Mycobacterium tuberculosis complex continues

这个谱系的进化
by SNPs and Indels

由单核苷酸多态性（SNP）
which are insertions or deletions as I showed you

和插入或缺失（Indels）延续
during the deletion analyses slide

结果产生不同的人类和动物谱系
leading to different human and animal lineages.

结核分枝杆菌复合群的动物谱系
And animal lineages of M. tuberculosis complex

起源于类非洲分枝杆菌谱系
originate from Mycobacterium africanum-like lineages.

所以 动物谱系不是人类菌株的起源
So, the animal lineages are not at the origin of human strains.

感谢您的关注
Thank you for your attention.